Rozprawy doktorskie na temat „Gellan gum”

The composition of gellan gum elaborated by wild type Sphingomonas paucimobilis was studied. Gas chromatographic methods of analysis were compared. Analysis of neutral sugars was carried out by Saemen hydrolysis and Stones method. Stones method was preferred due to its simplicity and the production of comparable results to those obtained following Saemen hydrolysis. The neutral sugars of gellan gum were analysed by GC/MS following preparation of per-methylated alditol acetates (PMAAs). Two methods were used for making alditol acetates, the Hakamori and sodium hydroxide (NaOH) methods. Although the NaOH method was simpler and faster to carry out, the results were not representative of the true composition of gellan gum. The Hakamori method revealed the presence of 33%, 1,4 rha, 30% 1,3 glc and 31% 1,4 glc in agreement with the sugar compositions of O'Neill et al. (1983) and Jansson et al. (1983) who elucidated the structure of gellan gum simultaneously. To determine the glucuronic acid content of gellan gum, samples were carboxy-reduced prior to analysis. Two methods were compared, the lithium aluminium deuteride and tetrahydrofuran (THF) methods. The THF method revealed the presence of 24%, 1, 4 rha, 24% 1,3 glc 26% 1,4 glc and 25% 1,4, 6 glc (corresponding to glcA). Physiological studies were carried out with twenty mutant strains of S. paucimobilis in batch culture. The chemical composition, viscosity, gelation and rheological properties of EPS produced from many of these strains were further investigated. Some of the strains elaborated polysaccharide, named fine precipitate, which differed chemically from gellan gum. Rheological measurements were carried out on several EPS samples using a mechanical rheometer. Large differences in gel strength were detected.

This research shows, for the first time, the freeze dried low acyl (LA) gellan gum, high acyl (HA) and HA:LA mixture gel structures and their properties upon rehydration. The water interaction with the gel structure is affected by the presence of acyl groups along the HA gellan gum polymer chain. The rehydration rate was shown to be lower for HA gellan gum and was not dependent on the polymer concentration. In the second instance, the effect of the gel pH on the freeze- and oven-dried structure is studied as well as the water re-absorption extent and rate. In this part, two drying methods, freeze- and oven-drying, were performed. Finally, a comparison of the three drying processes (freeze-, oven- and supercritical-fluid drying) is reported, proposing the effect on the molecular structure by investigation of the disordered chain domains of the gel network. As supercritical-fluid drying requires a pre-treatment in alcohol, its effect on the gellan gum network was investigated. It is shown how alcohols change the gellan gum configuration, leading to a considerable variation in gel texture. A novel supercritical-fluid batch rig is also proposed, characterised by high process flexibility and reduced set-up and energetic costs, particularly suitable for small/laboratory-scale use.

Gelation of polysaccharides under shear conditions results in the formation of a weak gel which is able to resist elastic mechanical deformation at small strains but will flow if subjected to higher strains. The resulting material, described in the literature as a fluid gel or a sheared gel, consists of gelled microparticles which can be formulated to collectively act in bulk, as pourable viscoelastic fluids whilst retaining true gel characteristics at the micro/nano level. The tuneable behaviour of these fluid gel systems makes them potentially useful in pharmaceutical applications. Fluid gels prepared from gellan gum are particularly attractive, due to its sensitivity to physiological fluids, unique rheological and physical properties, and current regulatory approval for use as a food additive and pharmaceutical excipient. Therefore, the aim of the present study was to investigate gellan gum fluid gels as a new modified release drug delivery platform. The formation and production of fluid gels using low acyl (LA) gellan, high acyl (HA) gellan and LA HA gellan blends was investigated and applied in three different dosage forms; a modified release oral liquid, a mucoadhesive nasal spray and a topical formulation. A modified release oral liquid was designed using a fluid gel prepared from LA gellan gum. It was demonstrated that 0.75 % w/w LA gellan gum fluid gel, containing ibuprofen as the drug, could be formulated to have a similar viscosity profile as a marketed oral ibuprofen liquid. Furthermore, due to the acid insolubility of gels prepared from LA gellan, no ibuprofen was released in stimulated gastric fluid. Subsequent release at pH 7.4 however, was affected by the duration of exposure and strength of the acidic pH used and a linear relationship between onset of release and the preceding duration of acid exposure was observed. Delayed release was a result of increasing gel stiffness, a consequence of the acidity of the initial release media and exposure time. A much faster release rate was measured when exposure time in acid was 10 min compared with 60 min. This study highlights the potential to design fluid gels that are tuned to have a specified stiffness at a particular pH and exposure time allowing the intelligent design oral liquids with specific modified release behaviour. The second part of this study was to prepare mucoadhesive nasal drug delivery systems to enhance the retention of the nasal spray dosage form in the nasal cavity. Several groups have investigated using LA gellan solution as a drug delivery vehicle but only limited research however, has been performed on HA gellan for this purpose, despite its properties being more conducive to mucoadhesion. HA gellan (even with low concentration 0.25 % w/w) produces highly elastic gels below 60 °C which make it difficult to spray using a mechanical spray device. To address this problem, fluid gels were prepared as these systems can behave as sprayable viscoelastic fluids. In this study the rheological behaviour was investigated and the mucoadhesion behaviour of fluid gels prepared from the two different types of gellan (HA and LA) and fluid gels prepared from a blend of LA HA gellan. The results demonstrated that by preparing fluid gels from a blend of LA HA gellan, the rheological properties were sufficient to spray through a standard nasal spray device. Moreover, the fluid gels significantly enhanced both HA and LA gellan mucoadhesion properties. In the final part of this thesis the topical application of gellan fluid gels was explored. A range of gellan fluid gel formulations were prepared containing diclofenac sodium for topical application. The rheological results showed that it was possible to produce a topical formulation with a viscosity and the mechanical strength similar to that of the commercially available Voltaren® gel using 1 % w/w of a 50:50 LA HA gellan blend. The permeation results highlighted that the penetration of diclofenac through procaine tissue is significantly increased by increasing gellan concentration and decreasing sodium ion concentration in the formulation.

Orientador: Rosiane Lopes da Cunha
Texto em português e inglês
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: As nano partículas possuem grande potencial para a liberação controlada de bioativos, porém ainda são pouco exploradas na área de alimentos. Neste trabalho foi estudada a formação de nanopartículas a partir da autoagregação de surfactantes, associação surfactante-polissacarídeo e complexação eletrostática entre diferentes polissacarídeos, no caso, quitosana e gelana. A compreensão das interações moleculares responsáveis pela formação das partículas e o conhecimento das variáveis que afetam sua formação permitem predizer e controlar suas propriedades. Tais interações dependem fortemente das características de cada macromolécula, como flexibilidade, estado conformacional e densidade de cargas que são diretamente afetadas pelas condições físico-químicas do meio como pH, força iônica e temperatura. Por isso, este trabalho foi dividido em três etapas. (I) Inicialmente foi avaliado o comportamento em solução dos polissacarídeos utilizados posteriormente para a formação de complexos. Os efeitos do pH e da temperatura nas características reológicas e no estado conformacional de soluções puras de gelana e quitosana foram estudados. A agregação da gelana foi mais sensível às alterações do meio que a quitosana. (II) Na segunda etapa, nanopartículas foram formadas por autoassociação de polissorbatos na presença de quitosana. A influência do comprimento da cauda hidrofóbica do surfactante e do pH do meio nas propriedades das partículas foi estudada por espalhamento de luz, reologia, condutivimetria e microscopia de luz polarizada. O tamanho e estrutura das partículas formadas pelo surfactante de menor cadeia hidrofóbica foram mais favoráveis à associação com a quitosana. O pH do meio (3,0 ou 6,7) não influenciou de maneira significativa as características das partículas. O efeito da concentração de quitosana na estrutura e tamanho de partículas foi analisado. Maiores concentrações levaram a viscosidades mais elevadas, impedindo a agregação das micelas e formando partículas menores. (III) No terceiro estudo, nanopartículas foram obtidas pela complexação eletrostática de gelana e quitosana. Os efeitos da razão de concentração de cada polissacarídeo, do tempo de estocagem a 25 °C e da presença de um surfactante nãoiônico (polissorbato) no tamanho, carga e quantidade de partículas formadas foram avaliados. Devido à menor densidade de carga e flexibilidade da gelana, maior quantidade deste polissacarídeo foi necessária para obtenção de partículas neutras. De forma geral, as partículas apresentaram aumento de tamanho ao longo das primeiras 100 horas após o preparo e não foram observadas mudanças significativas das propriedades das partículas devido à adição de surfactante. O método de preparo das amostras também foi estudado. Partículas preparadas pela mistura das soluções de polissacarídeos em dois passos foram consideravelmente maiores que as preparadas pela mistura em uma única etapa. Este trabalho confirmou a possibilidade de formação de nanopartículas promissoras para a encapsulação de bioativos em alimentos a partir da associação de biopolímeros e surfactantes, cujas propriedades poderiam ser moduladas em função da composição e condições de processo
Abstract: Nanoparticles are promising vehicles for bioactive delivery, but their potential has not been fully explored by the food industry. This work studied the formation of nanoparticles by self-assembly of surfactants, polysaccharide-surfactant association, and electrostatic complexes formed by different polysaccharides, especially chitosan and gellan gum. The knowledge of molecular interactions and the variables that affect particle formation allows predicting and controlling the properties of nanoparticles. These interactions depend on the characteristics of each macromolecule such as conformation, charge density and flexibility, which are affected by the physicol-chemical properties of the solution, such as pH, ionic strength and temperature. This work was divided in three parts: (I) Firstly it was studied the behaviour of each polysaccharide alone. The influence of the pH and temperature on the rheological properties and structural conformation of the pure gellan and chitosan samples was determined. Gellan aggregation was more strongly affected by such variables than chitosan. (II) In the second part, nanoparticles were obtained by polysorbate-chitosan association. The effect of the length of surfactant tail and the solution pH on the particle properties was studied by dynamic light scattering, rheological and conductivity measurements and polarizing microscopy. The size and structure of nanoparticles composed by the shorter surfactant were more appropriated to chitosan assembly. The pH (6.7 or 3.0) did not affect significantly the particle properties. The effects of chitosan concentration on particle structure and size were studied. Greater chitosan concentration led to smaller particles due to the increase in viscosity values which prevented micelles aggregation. (III) In the third study nanoparticles were produced by electrostatic complexation of chitosan and gellan gum. Particle size, charge density, stability and complexes number were evaluated as a function of polysaccharide concentration, chitosan:gellan ratio and the presence of a non-ionic surfactant. Due to the stiffness and low charge density of gellan gum, a greater amount of such polysaccharide was necessary to obtain neutral particles. Overall particles showed an increase in size during 100 hours of storage at 25 °C, but no significant changes on particle properties were observed due to surfactant addition. The methodology of particle preparation was also evaluated. Particles prepared by 2 mixing steps were markedly larger than those prepared by mixing polysaccharides in a single step (all together). This work showed that it is possible to produce nanoparticles with promising application on bioactive delivery by biopolymer-surfactant association, since their properties could be modulated as a function of composition and process conditions
Doutorado
Engenharia de Alimentos
Doutor em Engenharia de Alimentos

O objetivo deste trabalho foi desenvolver formulações de pellets contendo teofilina, utilizando a goma gelana como aglutinante. Inicialmente, o uso desse polímero foi avaliado em comparação com o PVP. Por meio de delineamento experimental 32 foram analisadas as variáveis aglutinante e diluente. Os pellets obtidos apresentaram resultados satisfatórios na friabilidade e esfericidade. Verificou-se que o diluente exerceu influência significativa na granulometria, de modo que formulações com manitol apresentaram maior formação de pó após a produção. Com relação ao aglutinante sua maior influência foi na dissolução, pois formulações com gelana apresentaram eficiência de dissolução, em média, menor que as formulações com PVP. A seguir, foi avaliada a influência do polímero, sua quantidade, e seu modo de incorporação nas formulações. Para isso foi realizado um delineamento experimental 23, tendo a gelana, a quantidade, e a incorporação como variáveis. Verificou-se que a característica que sofreu maior influência das variáveis foi a granumoletria, pois as condições empregadas influenciaram significativamente o rendimento de pellets. Com relação à friabilidade, esfericidade e dissolução, não foram observadas diferenças significativas entre os resultados, ainda que algumas tendências puderam ser identificadas.
Aim of this work was to develop pellet formulations containing theophylline, using gellan gum as a binding agent. Inicially, application of the polymer was compared with PVP. By a 32 experimental design, variables binder and diluent were analyzed. Pellets presented satisfactory results in friability and sphericity. It was found that the diluent exerted significant influence on granulometry, so formulations with mannitol showed higher dust formation after production. Regarding binder variable, its higher influence was in the dissolution, since formulations with gellan presented smaller dissolution efficiency than formulations with PVP. Next, the influence of polymer, amount, and incorporation were evaluated. In this part, a 23 experimental design was carried out, with gellan, quantity, and incorporation as variables. It was found that granulometry was the most influenced by variables. So conditions employed influenced significantly the yield of pellets. Regarding friability, sphericity and dissolution, no significant differences were observed, although some trends could be identified.

A novel approach that may impact on satiety, whilst meeting the demands of consumers, is the use of hydrocolloids that respond to the environment (acidic) conditions inside the human stomach by self-structuring. This thesis seeks to investigate the in vitro acid-induced gelation (“structuring”) of the mixed biopolymer systems; low-acyl and high-acyl gellan gum, and low-acyl gellan fluid gels. To explore this concept, a variety of acid structures were obtained, which were characterised by texture analysis, rheology and dynamic scanning calorimetry. The gel structures were found to rely on the pH, hydrocolloid concentration, percentage weight of each hydrocolloid used and the processing conditions used during their production. It is suggested that the use of gel alone is more than capable of providing prolonged satiety but leads to unpleasant sensations for the consumer if there is no delivery of energy to the body to compliment the sensation of satiety. Materials should be included that will modulate the energy delivery and slowly release calories over time. This research shows that the addition of co-solutes such as sugar and the measurement of their subsequent release from hydrocolloid gels could provide a first step to tackling these issues.

Orientador: Rosiane Lopes Cunha da
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A influência da concentração de polissacarídeo, do pH de equilíbrio (de 2,0 a 4,0) e da aplicação de tratamento térmico (70 ou 90ºC/ 30min) sobre as propriedades reológicas e de microestrutura de géis de gelana acidificados lentamente pela adição de glucona-d-lactona (GDL) foi estudada nesse trabalho. Os sistemas foram caracterizados quanto à cinética de gelificação e propriedades dos géis formados, como as reológicas em compressão uni- e biaxial, capacidade de retenção de água (WHC), microestrutura e turbidez. Em todas as amostras que formaram géis auto-sustentáveis, com exceção daquelas com 1% de gelana nos menores pHs, foi possível determinar o ponto de gel a partir de ensaios oscilatórios, sendo que o tempo do mesmo diminuiu com o aumento da concentração de polímero, com o tratamento térmico, e com a diminuição do pH final. Todos os géis auto-sustentáveis foram transparentes e não apresentaram sinerese. A tensão de ruptura aumentou com a diminuição do pH, aumento da concentração de gelana e submissão a tratamento térmico antes da adição de GDL, porém diminuiu quando este foi realizado depois da acidificação. A WHC tornou-se maior com o aumento da concentração de gelana e do pH nos sistemas de concentração 1% e 0,5%, no entanto, a 0,2%, ela aumentou com a diminuição do pH. A microestrutura dos géis mostrou correlação direta com as propriedades reológicas em compressão. A análise da dimensão fractal dos géis só foi possível em pHs 2,5 e 2,0, e os sistemas apresentaram estrutura fractal com regime de ligação fraca. Todas as amostras mostraram comportamento de fluido lei da potência em escoamento extensional. A correlação entre turbidez e força dos géis foi linear. No entanto, a turbidez mostrou-se mais sensível às mudanças estruturais do que as propriedades mecânicas
Abstract: The influence of polysaccharide concentration, pH at equilibrium (2,0 - 4,0) and annealing (70 or 90ºC/30min) on rheological properties and microstructure of gellan gels formed by acidification of glucon-d-lacton GDL was evaluated in this study. Gels were characterized in relation to their gelling kinetics, mechanical properties at fracture, water holding capacity (WHC), microstructure, turbidity and rheological parameters in extension. In all samples, with exception to that of 1% concentration formed at pHs 2,5 and 2,0, it was possible to determine the gel point by oscilatory measurements. The time at which gel point occurred diminished by raising polymer concentration, annealing and lowering final pH. All gels were transparent and exhibited no sign of syneresis. Failure stress increased at lower pH values, higher concentrations and gels were even stronger when made from annealed solution. When gels were annealed after pH reached equilibrium, their failure stress decreased. The WHC for 1% and 0,5% gels was higher at enhanced concentrations and higher pH values and for 0,2% gels, it was higher at lower pH values. Microstructure of gels showed straight correlation with their rheological properties in compression. Analysis of fractal dimension of the gels was only possible at pHs 2,5 and 2,0. In this case, the systems showed fractal structure in the weak¿link regime. All samples showed power law behaviour in extension. The turbidity and the force of gels showed linear correlation. Nevertheless turbidity measurements were more sensitive to structural changes than were mechanical properties
Mestrado
Mestre em Engenharia de Alimentos

This Diploma thesis deals with optimization of the hydrogel preparation with various additions of substances affecting adhesion, studying the adhesion of prepared hydrogel systems and modifying their adhesion properties. Prepared hydrogels were agarose, alginate and gellan polymer systems with additions of acrylic acid, acrylamide, calcium chloride and Tween 20. The experimental part of the diploma thesis focuses first on optimizing the preparation of individual hydrogels, from which, according to certain criteria, suitable representatives were selected to study adhesion and its controlled modification. Agarose and gellan hydrogels were selected as suitable representatives, which were then used in rheological measurements. Based on these measurements, the effect of the adhesive force and the adhesive work of individual materials was evaluated. It was found that the best adhesion properties of the materials used are unadjusted hydrogels, in the case of hydrogels with the addition of another substance, a negative effect on adhesion was observed, when the adhesion force was reduced and also the adhesion work was reduced.

O tratamento farmacológico de patologias bucais é conduzido, geralmente, por via de adminis-tração local. No entanto, devido ao pouco tempo de permanência do fármaco no local de ação, esse tratamento pode ser bastante comprometido. Assim, este trabalho teve por objetivo o de-senvolvimento de formas farmacêuticas que proporcionem a liberação local de triancinolona na cavidade oral. Foram produzidos filmes e comprimidos mucoadesivos a partir de polímeros naturais como gelana e pectina. Os filmes bucais foram preparados por meio de evaporação do solvente (solvent casting) utilizando diferentes quantidades de polímeros. As matérias-primas e os filmes foram caracterizados fisico quimicamente utilizando espectroscopia vibracional (in-fravermelho com transformada de Fourier e Raman) e difração de raios X. As propiedades físicas e mecânicas dos filmes também foram avaliadas. Além disso, realizou-se os ensaios de mucoadesividade e de dissolução do fármaco. Os comprimidos foram preparados por com-pressão direta usando como base os polímeros naturais. Diferentes parâmetros em relação as misturas e as formulações foram avaliados tais como as propriedades de fluxo dos pós consti-tuintes, peso médio, dureza, friabilidade e desintegração. Em relação aos filmes bucais, estes foram obtidos com sucesso através de um método simples, sem a utilização de agentes reticu-lantes, ácidos ou solventes orgânicos. Todos apresentaram bons resultados nas propriedades avaliadas, no entanto as formulações com quantidades intermediarias de polímeros foram as melhores. Dentre as formulações de comprimidos preparadas, apenas 4 apresentaram boas ca-racterísticas, no entanto, os resultados dos ensaios de dissolução mostraram que estas formula-ções têm capacidade de agir como sistema de liberação controlada de fármacos.
Pharmacological treatment of oral pathologies is usually conducted by local administration. However, due to the short time the drug stays in the site of action, this treatment can be quite compromised. Thus, the objective of this work was to develop pharmaceutical forms that pro-vide the local release of triamcinolone in the oral cavity. Mucoadhesive films and tablets were made from natural polymers such as gellan and pectin. The buccal films were prepared by sol-vent casting using different amounts of polymers. The raw materials and films were characte-rized physically chemically using vibrational spectroscopy (FTIR and Raman) and X-ray diffraction. The physical and mechanical properties of the films were also evaluated. In addi-tion, the mucoadhesive and drug dissolution tests were performed. The tablets were prepared by direct pressing with the natural polymers. Different parameters in relation to mixtures and formulations were evaluated such as the flow properties of the constituent powders, average weight, hardness, friability and disintegration. In relation to oral films, these were successfully obtained by a simple method, without the use of crosslinking agents, acids or organic solvents. All presented good results in the evaluated properties, however the formulations with interme-diate amounts of polymers were the best. Among the tablet formulations prepared, only 4 sho-wed good characteristics, however, the dissolution test results showed that these formulations have the ability to act as a controlled drug delivery system.

Mestrado em Engenharia Alimentar - Processamento de Alimentos - Instituto Superior de Agronomia
The aim of this study was the analysis of volatile compounds responsible for the flavour of a texturized mango product, namely terpenes, lactones, aldehydes and alcohols, evaluating the impact of the processing applied: thermal treatment to inactivate enzymes, texturization with gellan gum, application of an edible coating and two conservation processes. The analytical method used was gas chromatography combined with the technique of solid phase microextraction. The product under development consists in a bar of mango puree texturized using the hydrocolloid gellan gum, matching 1% of the weight of the mango puree. This percentage is composed by gellan low and high acyl, with the ratio 25/75, respectively. The product also has an edible coating, comprised by 1% of gellan gum which is formed with a 90/10 low acyl/high acyl ratio. The thermal treatment to inactivate enzymes of mango puree caused a decrease of most volatile compounds. The texturized mango puree has exhibited an ability to release the volatiles, similar to mango puree. The edible coating applied to the surface of the texturized product showed retention capacity for a small number of volatiles. The applied conservation processes, high hydrostatic pressures and thermal treatments, showed similar effects and didn’t change the volatile profile of the bar significantly. Microbiologically the product proved to be stable and both conservation treatments applied were effective in microbiological control.

Selected food gums (hydrocolloids) were tested for their abilities to encapsulate enzymes for acclerating cheese ripening. The effect of pH and acidity on gel strength of the gums was determined. Enzyme was entrapped in k-carrageenan, gellan gum and milk fat and incorporated into cheese milk prior to cheese making. The cheese produced was tested for protein breakdown and amino acid production during ripening and textural and sensory properties of the ripened cheese were also evaluated. The findings and significance of this study and the literature review are presented. Gels were produced from alginate. Most gels showed reduced strength after treatment in solutions of either modified acidities or pH. There was, however, no significant change in gel strength between the different treatments for most of the gums. The activity of encapsulated enzyme were also investigated. Enzymes encapsulated in k-carrageenan, gellan and alginate gums retained higher activities than the other gums studied. Enzymes entrapped in agar gels had the least retention of activity. The retention of enzyme capsules produced from gellan, k-carrageenan and milk fat in cheese curd was investigated. Loss of encapsulated enzymes in cheese whey was also determined. Enzyme loss in the whey ranged from 5.6 to 17.9% with the highest losses observed with milk fat and the least with gellan gum capsules. Most of the cheeses treated with enzyme capsules showed higher levels of amino acid within two weeks than control cheese. After two weeks, all experimental cheeses showed higher production of amino acids than the control cheese. The addition of enzyme capsule to cheese did lead to a higher growth level of microorganisms. The experimental cheese exhibited lower score than the control cheese for most textural properties. The experimental cheeses were not significantly different in flavour and aroma from the control cheese. K-carrageenan treated cheeses were recorded as having the highest score for bitter after taste. Except for those cheeses treated with k-carrageenan capsules, the overall acceptability for the trial cheeses were not significantly different from that of 6 month old untreated cheese.
Master of Science (Hons)

Magnetic nanoparticles (MNPs) have found use in a wide variety of biomedical applications including hyperthermia, imaging and drug delivery. Certain physical properties, such as the ability to generate heat in response to an alternating magnetic field, make these structures ideal for such purposes. This study's objective was to elucidate the mechanisms primarily responsible for RF MNP heating and determine how such processes affect polymer solutions that might be useful in drug delivery. 15-20 nm magnetite (Fe3O4) nanoparticles at 0.2% and 0.5% concentrations were heated with RF fields of different strengths (200 Oe, 400 Oe and 600 Oe) in water and in 0.5% gellan gum solution. Mixing and fan cooling were used in an attempt to improve accuracy of data collection. Specific absorption rate (SAR) values were determined experimentally for each combination of solvent, concentration and field strength. Theoretical calculation of SAR was performed using a model based on linear response theory. Mixing yielded greater precision in experimental determination of SAR while the effects of cooling on this parameter were negligible. Solutions with gellan gum displayed smoother heating over time but no significant changes in SAR values. This was attributed to low polymer concentration and lack of structural phase transition. The LRT model was found to be adequate for calculating SAR at low polymer concentration and was useful in identifying Neel relaxation as the dominant heating process. Heating trials with MNPs in 2% agar confirmed Neel relaxation to be primarily responsible for heat generation in the particles studied.

Drug delivery to the nasal and ocular mucosa faces several obstacles. One of these is from the effective clearance mechanisms present in the nose and eye. Polymer gels with suitable rheological properties can facilitate the absorption of poorly absorbed drugs by increasing the contact time of the drug with the mucosa. This has been attributed to the rheological and mucoadhesive properties of the gel. The main objective of this thesis was to investigate the importance of these features for the anticipated in vivo contact time, here exemplified by the ocular and nasal routes of administration.

The in situ gelling polymer gellan gum was found to have a favourable rheological and in vivo performance. When administered in the nasal cavity of rats, a gel was formed that could remain at the site of administration for up to 4 hours. In addition, the epithelial uptake and transfer of a 3 kDa fluorescein dextran was higher than for a mannitol solution. Therefore, it was concluded that a gellan gum formulation should be a promising strategy for nasal drug delivery.

The potential mucoadhesive properties of a variety of polymer gels were investigated using a rheological method and by measuring the tensile force required to detach the gel from a mucosa. With both methods the rheological properties of the gel were a determining factor for the results obtained. The rheological method was found to have several limitations. One of these was that a positive response, interpreted as mucoadhesion, was only seen with weak gels. The tensile method could, in contrast, detect strengthening of the mucus only for strong gels. However, this method reflects the in vivo performance of the gel better than the rheological method.

Finally, dielectric spectroscopy was explored as a tool for investigating the likelihood of intimate surface contact between the gel and the mucus layer. This novel approach involved determining the ease with which a charged particle can pass the gel-mucus interface layer, and may enable the study of the events at the interface closer to the molecular level, than is possible with the rheological and tensile strength methods.

Orientador: Rosiane Lopes da Cunha
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Tendo em vista a necessidade do entendimento das interações entre ingredientes para o desenvolvimento de novos produtos lácteos e para o controle e manipulação de suas características de estabilidade e textura, este trabalho teve como objetivo elucidar as interações entre as proteínas do leite e a goma gelana. Foi estudado o efeito de diferentes concentrações de gelana, caseinato de sódio e concentrado protéico de soro de leite, assim como o estado conformacional do polissacarídeo, nas propriedades de textura, microestrutura e estabilidade em relação à perda de água dos sistemas formados. A transição conformacional da gelana em diversos valores de pH foi estudada por ensaios reológicos oscilatórios a baixas freqüências. Foi observado que a transição de estado desordenado ¿ ordenado do polissacarídeo é irreversível com a temperatura em pH 3,5, enquanto em maiores valores de pH os géis formados são termorreversíveis. Além disso, sistemas em pH 3,5 apresentaram viscosidade e caráter elástico mais pronunciados, enquanto que na faixa de pH de 5,3 a 7,0, não foram observadas mudanças significativas nas características reológicas do material. Em sistemas mistos proteínas-polissacarídeos, diferentes estruturas foram observadas de acordo com as concentrações de biopolímeros e o estado conformacional do polissacarídeo, resultando em alterações das propriedades mecânicas, de retenção de água e de solubilidade dos sistemas. Sistemas bipoliméricos caseinato de sódio ¿ gelana formaram coacervados em altas concentrações de proteína e apresentaram microestrutura compacta, refletindo em maiores valores de tensão de ruptura, elasticidade e baixa capacidade de retenção de água. Já nos sistemas tripoliméricos, não foram observados coacervados devido ao favorecimento das interações entre as proteínas que ocasionou um aumento dos valores de capacidade de retenção de água das amostras. A gelana e as proteínas do soro apresentaram incompatibilidade termodinâmica em altas concentrações poliméricas, confirmada pelos ensaios de solubilidade protéica em água, levando a formação de sistemas mais frágeis, menos deformáveis e com uma estrutura mais porosa, que contribuiu para o aumento da capacidade de retenção de água e redução da sinerese dos mesmos. O uso de gelana em estado desordenado no preparo das amostras com alta concentração protéica induziu a formação de complexos eletrostáticos entre as proteínas e as moléculas de gelana individuais, diminuindo a rigidez dos sistemas
Abstract: As the ingredient interactions are important for the development of new milk products and for the control and management of its stability and texture properties, the aim of this work was to elucidate the milk proteins and gellan gum interactions. The effect of different concentrations of gellan gum, sodium caseinate and whey protein concentrate, as well as the polysaccharide conformational transition on the systems texture properties, microstructure and water release were studied. The gellan gum conformational transition was studied in different pH values by oscillatory shear measurements at low frequencies. It was observed that the conformational polysaccharide transition (coil-helix) was thermoirreversible at pH 3.5 while at higher pH values the gels were temperature reversible. Moreover, systems at pH 3.5 showed higher viscosity and storage modulus, but at 5.3 ¿ 7.0 pH range it was not observed significant differences on the material rheological properties. Protein-polysaccharide systems showed different structures according to the polymers concentration and polysaccharide conformation, resulting on variations of its mechanical properties, water binding and solubility. Bi-polymeric systems composed of caseinate and gellan formed coacervates at high protein concentration leading to a compact structure which reflected in higher rupture stress, elasticity and water holding capacity. Nevertheless, on multipolymeric samples it was not observed coacervates probably due the enhance of protein-protein interactions that lead to increase on water holding capacity values. Gellan gum and whey proteins showed thermodynamic incompatibility at high polymer concentrations, confirmed by the solubility measurements, and induced the formation of weaker and less deformable gels, which had a porous structure with more ability to hold water. The use of coil gellan on the preparation of the samples with high protein concentration resulted on electrostatic complexes formation between the proteins and the individual gellan molecules, reducing the gels hardness
Mestrado
Mestre em Engenharia de Alimentos

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia
The aim of this work was the development of an appealing fresh fruit translucent candy, with gelling agents, alternative to the natural fruit pectin. Three fruit gel formulations were developed, named Fresh Fruits Marbles (FFM), two apple based and one lemon juice based. These were inspired on the traditional French patisserie products Patê de Fruits. At first, high methoxyl (HM) pectin (1.5%) was used as a gelling agent and at second a mixture of iota carrageenan/locust bean gum (1.5% /1.5 %) were used. The edible coatings were sucrose, olive oil and HM citrus pectin coating (3%). HM pectin (2%) was used in the formulation of gel translucent. The coatings were: HM pectin (3%), carboxymethyl cellulose (1%) and iota carrageenan (1%). The characterization analysis was focused on chemical (pH, humidity, aw, total soluble solids), physical (texture and color) and microbiologic characteristics (mold and yeast counting). The iota carrageenan/locust bean gum mix, in the gel on the Apple based FFM, promoted a more elastic texture. The sugar coating is the one that best preserves the FFM. Pectin HM (3%) coating on lemon juice based FFM promoted an increase in hardness. All developed FFM looked and tasted well – a promising product.

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais
The use of hydrogels as platforms for Tissue Engineering and Regenerative Medicine approaches has been growing in the past years. Their resemblance to the natural extracellular matrix, and facility to modify and tailor their final properties are some of the aspects that appeal researchers around the world to use this type of material. Different hydrogel sources have been exploited and processed using different methodologies, resulting in different structures that can be tailored for the intended application. Indeed, cell-material interactions can be adjusted by using distinct hydrogel designs, processing methods or sources. One of the natural materials that has been considered for Tissue Engineering and Regenerative Medicine strategies is gellan gum. This natural polymer has been used for different applications over the last years, on its natural form or after chemical modifications. Among such modifications, gellan gum methacrylation already showed promising results on Tissue Engineering. Nevertheless, the impact of the crosslinking and processing strategies used with the material are still not well established. This thesis aimed to highlight the versatility of this polymer, achieved by using different crosslinking strategies. Ionic crosslinking with calcium chloride, one of the most used methods to prepare gellan gum hydrogels, were firstly considered envisioning bone tissue engineering. Ionic crosslinking was also used to prepare traceable hydrogels, taking advantage of the affinity of methacrylated gellan gum to the divalent paramagnetic manganese ion. The ionic-responsive property of gellan gum allowed a minimally invasive administration of gellan gum in the vicinities of the central nervous system, as well as the desired traceability. At last, a different strategy was used to obtain hydrogels with immunoprotective capabilities, intended to be used on immunoisolation strategies. Polyelectrolyte complexation of gellan gum with poly l-lysine yielded a semipermeable membrane, capable of blocking the passage of the immune cells towards the hydrogel core, where therapeutic cells are encapsulated. Altogether, the different approaches used on the scope of this thesis showed the versatility of methacrylated gellan gum and the importance of carefully chose the crosslinked methodology to prepare hydrogels, as they greatly influence final hydrogel behaviour.
O uso de hidrogéis como plataformas para a Engenharia de Tecidos e Medicina Regenerativa tem vindo a crescer nos últimos anos. A semelhança com a matriz extracelular natural, assim como a facilidade em modificar e adaptar as suas propriedades finais, são alguns dos fatores que levam à utilização deste tipo de material. Hidrogéis provenientes de diferentes fontes foram já explorados, e processados usando diferentes metodologias, resultando em diferentes estruturas que podem ser personalizadas para a aplicação pretendida. De facto, as interações entre células e materiais podem ser ajustadas usando hidrogéis preparados com diferentes métodos ou formas de processamento. Um dos materiais naturais considerados para as estratégias de Engenharia de Tecidos e Medicina Regenerativa é a goma gelana. Este polímero natural tem sido utilizado para diferentes aplicações ao longo dos últimos anos, na sua forma natural ou após modificações químicas. Entre essas modificações, a metacrilação da goma gelana já apresentou resultados promissores em Engenharia de Tecidos. No entanto, o impacto das estratégias de reticulação e processamento usadas no material, ainda não está bem estabelecido. Esta tese teve como objetivo destacar a versatilidade deste polímero, pelo uso de diferentes estratégias de reticulação. Inicialmente, foi considerada a reticulação iónica com cloreto de cálcio, um dos métodos mais utilizados para a preparação de hidrogéis de goma gelana, para aplicação em regeneração óssea. A reticulação iónica foi também usada para preparar hidrogéis rastreáveis, aproveitando a afinidade da goma de gelana metacrilada com o manganês, um ião divalente paramagnético. A reticulação da goma gelana na presença de iões permitiu uma administração minimamente invasiva do material nas proximidades do sistema nervoso central, bem como a rastreabilidade desejada. Finalmente, uma estratégia diferente foi usada para obter hidrogéis com capacidade imunoprotetora, destinados a ser usados como immunoproteção. Complexos de polieletrólitos formados pela interação da goma de gelana com poli-L lisina originaram uma membrana semipermeável, capaz de bloquear a passagem das células do sistema imunitário em direção ao núcleo de hidrogel, onde as células com fins terapêuticos são encapsuladas. Em suma, as diferentes abordagens utilizadas no contexto desta tese mostraram a versatilidade da goma de gelana metacrilada e a importância de escolher cuidadosamente a metodologia de reticulação para preparar hidrogéis, pois estas têm grande influência no comportamento final do hidrogel.

碩士
東海大學
化學工程與材料工程學系
107
Gellan gum (GG) is proved with excellent biocompatibility as well as hydrophilic properties and is widely used in clinical. However, the mechanical strength of pure film is still an insurmountable shortage especially under simulating body fluidic conditions. In order to face the challenge at the application, this study takes advantage of modified material, gamma-polyglutamic acid (γ-PGA) with calcium as cross-linker which can be formd tri-dimension structure significantly enchancing the stress to improve the mechanical properties. Nonetheless, the producing membrane by the cross-linking mechanism is too brittle to be accepted in clinical. Thus, glycerol is used to tremendously advance the elongation and improve the brittleness. In this way, we can fabricate the composite membrane as regenerate membrane with promising material properties and practicability in bone tissue engineering. The concept of this study is to adjust the γ-PGA and glycerol ratio under fixed gel concentration. We attest the material properties, biocompatibility and cell proliferation (MG63) to prove the feasibility in bone tissue regeneration. The water content test shows that the composite film has the superior water retention that can trap the water inside the gel. According to the tensile test, DSC and AFM analysis, the results display that γ-PGA molecules can boost the original mechanical properties and the crystallization behavior because of physical cross-linking mechanism. In addition, the results represent that appropriate ratio for cross-linking can effectively sustain the structure. Tensile test also shows that inclusion of glycerol into composite membrane improves the strain-at-break at a cost of reducing the tensile strength that is beneficial for operating. By the examination of degradation test, the composite membrane can surely endure for an extended period of time fulfilling the short-term clinical requirements. In the other hand, biocompatibility tests claim that the composite film demonstrates high protein adsorption and excellent cell compatibility. The results also show that cell proliferation does not cause negative effect when the membrane further blend with glycerol. The evidences represent that advancing the ductility may not affect the cellular behavior. It could further overcome the handling problem in operating. In the end, the section discusses the applicability in bone tissue engineering by MG63. The result represents that the composite membrane with basic mechanical strength can successfully promote bone cell proliferation. Following in culture, the result displays that there is no abnormal secretion of alkaline phosphatase enzyme, and enzyme activity is directly proportional to the strength of the membrane. Following the mineralization test, the composite membrane satisfies the essential requirements of bone tissue regeneration, during which can effectively guide bone cell proliferation and induce calcification. Meanwhile, the result also strongly correlates with tensile strength. Therefore, it is an indispensable prerequisite for bone tissue regeneration that substrates with sustainable structure can increase the productibity of tissue regeneration.

碩士
大同大學
材料工程學系(所)
100
In this study, we use the low-temperature plasma modification technology to deposite thin film or activate the substrate surface. Explore the impact of plasma treatment on fixing Gellan gum.The water contact angle showed that after IPA plasma treatment the contact angle was improved to 60~70 degree, and after oxygen plasma treatment the contact angle was improved less than 10 degree. and oxygen plasma treatment can let surface connect with peroxide functional groups. The surface properties of TPU, glass, Al thin film were also characterized by electron spectroscopy for chemical analysis and scanning electron microscope. By the AFM images can seen that the roughness has increased after oxygen plasma treated. Because gellan gum has strong cohesion it is hard to get a uniform film on the surface, therefore, increase the surface hydrophilicity can decrease the contact angle of gellan gum, then we can get good combination of gellan gum with the substrate and uniform coating thin film on the surface. With the cell culture can seem IPA plasma can't effectively improve the combination of substrate and gellan gum but can effectively increase the cell-attached later via cell culture experiments.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019
O síndrome do olho seco (DES) é um dos distúrbios oculares mais comuns para os quais o tratamento combinado inclui a modificação do ambiente ocular e administração tópica de lágrimas artificiais. A ciclosporina A (CyA), um agente imunossupressor, tem sido o mais promissor tratamento para o DES. No entanto, os sistemas atualmente disponíveis que utilizam óleos para fornecer CyA topicamente são pouco tolerados, induzindo dor na instilação da gota e subsequente descontinuação do tratamento, para além de apresentarem uma baixa biodisponibilidade. No presente estudo, uma nova abordagem tecnológica foi aplicada para melhorar o tempo de permanência do CyA no fluido lacrimal após a instilação da formulação como colírio. A associação de um sistema de nanotecnologia constituído por tensioativos não-iónicos, nanomicelas de auto-montagem e polímeros gelificantes in situ, como goma gelamica, foi desenvolvida tirando proveito da capacidade das nanomicelas para melhorar a solubilidade do CyA, e da capacidade do hidrogel para aumentar o tempo de residência do fármaco na zona precorneal. Pretende-se desta forma promover a entrega de CyA através de alvos oculares externos/internos, aumentando sua biodisponibilidade ocular. A formulação preparada foi caracterizada, no que diz respeito às suas propriedades físico- químicas, estabilidade química, concentração micelar crítica, citotoxicidade e farmacocinética. Os resultados mostraram que as formulações preparadas exibiam propriedades físico- químicas aceitáveis, com um pequeno tamanho de partícula e uma alta eficiência de aprisionamento do fármaco. Os estudos de estabilidade indicaram que não ocorreu degradação química durante o tempo de armazenamento nas duas temperaturas analisadas. Foi também avaliada experimentalmente a concentração micelar, crítica cujos resultados foram confirmados pela literatura e calculados usando a equação de fluxo. Os estudos de citotoxidade mostraram que as formulações desenvolvidas não são citotóxicas, confirmando a inocuidade dos tensioativos utilizados neste estudo. 7 O perfil farmacocinético mostrou que as formulações aumentaram o tempo de semi-vida e a AUC (área debaixo da curva), Ke (constante de eliminação) mostrou-se ligeiramente menor em relação à formulação de referência. Além disso, os sistemas de gelificação in situ foram muito bem tolerados, sem fenómenos de irritação visível, danos oculares ou sinais clínicos anormais na córnea. Este estudo resultou no desenvolvimento de um novo sistema de administração de CyA pela via ocular, o qual permitiu melhorar a sua solubilidade deste fármaco, bem como a sua biodisponibilidade após a sua aplicação ocular.
Dry eye syndrome (DES) is one of the most common disorders of the eye for which combined treatments include the modification of the ocular environment and topically administered artificial tear substitutes. Cyclosporine A (CyA), an immunosuppressive agent, has long been the mainstay treatment for DES. However, the currently available systems using oils to deliver CyA topically are poorly tolerated inducing pain on drop instillation and subsequent discontinuation of treatment, besides providing a low bioavailability. In the present study, a new technological approach has been applied to improve the residence time of CyA in the tear fluid after instillation of the formulation as eye drop. A association of a nanotechnology system constituted by non-ionic surfactants, self- assembling nanomicelles and in situ gelling polymers, as gellan gum, has been developed by taking advantage from nanomicelles ability to improve the CyA solubility and the hydrogel capacity to increase drug precorneal resident time, with the purpose of promoting CyA deliver through outer/inner ocular targets to increase its ocular bioavailability. The prepared formulations were characterized with regard to its physicochemical properties, chemical stability, critical micellar concentration, cytotoxity and pharmacokinetic. Results showed that the prepared formulations exhibited acceptable physical properties, with a small particle size and a high entrapment efficiency. The stability studies indicated that no chemical degradation occurred during the storage time at the two analysed temperature. It was also experimentally evaluated the critical micellar concentration, whose results were confirmed by the literature and calculated using the flowing equation. The cytotoxity studies showed that the developed formulations are not cytotoxic, confirming the ocular safety of the surfactants used for this study. The pharmacokinetics profile showed that the formulations produced a higher half-life and AUC (area under the curve), and slightly lowers Ke (elimination constant) with respect to the reference formulation. Moreover, the in situ gelling systems were very well tolerated by the animal, without any visible irritation phenomena neither ocular damage nor abnormal clinical 9 signs to the cornea. This study proposed the development of a new ocular administration system of CyA, improving the solubility of the drug, as well as its bioavailability after ocular application.
Hospital Garcia de Orta e Farmácia Higiene Montijo

Este trabalho foi efectuado com o apoio da Universidade de Lisboa, Instituto Superior de Agronomia com o Centro de Engenharia dos Biossistemas (CEER
The development of novel food products designed to maximise both the health benefits and enjoyment of the consumer’s eating experience is an important area of research nowadays. Fruits and vegetables are also increasingly recognized as important components of a healthy diet. Within this context, the work in this thesis aimed the development of a novel convenient food product – fresh like mango bars - based on mango (Mangifera indica L) and gellan gum, designed to have sensory quality similar to that of fresh mango, to be of easy consumption and to have an extended shelf-life. The influence of gellan concentration and of the ratio of L (low acyl)/H (high acyl) gellan on the rheological and microstructural properties of mango/gellan gels was studied. This allowed optimising the concentration of gellan. The effect of the L/H ratio on texture, microstructure, syneresis and sensory acceptance was evaluated. Results separated bars into two sets: one showing a higher hardness and lower syneresis (only L gellan and L/H in the ratios of 75/25 and 50/50); and the other with bars having a higher proportion of H, with a softer structure with higher cohesiveness and syneresis (L/H25/75 and only H gellan). Through sensory analysis the bar with L/H 25/75 was identified as the preferred one. An edible coating was developed to improve the characteristics of the product. Its optimization was performed by the use of experimental design and mathematical modelling. Results showed that the coating improved appearance and firmness of bars, and reduced syneresis and volatiles loss during storage. Thermal and high hydrostatic pressure preservation treatments were applied on packaged products. Both treatments had similar performance considering microbial content and aroma release. Thermally treated bars were stored at 5 °C during 21 days, without changing significantly their texture, colour, microbial loading and sensory attributes. Overall results show that the process allows producing a stable ready-to-eat mango bar product appreciated by the sensory panellists.
Fundação para a Ciência e a Tecnologia (FCT)- (SFRH/BD/64341/2009); FCT-MCTES funding the projects PTDC/AGR-ALI/114706/2009 and PEst – OE/AGR/UI0245/2014

Higher separation efficiency and resolution have been of great interest in chromatography and have become increasingly important in recent years mainly driven by the challenges of either more complex samples or high sample quantity. Therefore, for the development of new chromatographic matrices is increasingly important to improve the purification efficiency and to decrease the use of resources. Gellan gum is a polysaccharide polymer with natural anionic nature and ability to form thermoreversible gels, seeming to have potential to be used as a chromatographic matrix. In the presence of cations, the gellan polymer suffers conformational transition accompanied by the formation of a three dimensional network, forming a gel. In this work, it was intended to prepare of a stable gellan gum gel to be used as a chromatographic matrix. In order to increase the stability of the gellan gels, different experimental conditions were tested. Experimental design was used to obtain optimal conditions for the gel stability. Due to negative charge of these gels, it was possible to study the interactions established with three model proteins (bovine seric albumin (BSA), α-chymotrypsin and lysozyme). Gellan gum was able to interact with two of these proteins, being able to elute them with an increase in the ionic strength. In this assays a MES buffer with pH 6.2 was utilized. This pH conferred negative charge to BSA and positive charge to α-chymotrypsin and lysozyme, due to their isoelectric points. Assays of dynamic binding capacity were performed to find more characteristics of this new matrix and comparing with commercial resins. The values of dynamic binding capacity of the gellan gum to 10% and 50% breakthrough were 3,9 mg/ml and 17,4 mg/ml, respectively. These values were similar to commercial resins. These results showed that gellan gum might be an innovative and promising chromatographic matrix due to its versatility to interact with different biomolecules and its gelling ability. Thus, the gellan gum gel could have potential to be applied in different scientific domains (purification of complexes cellular extracts or nucleic acids).
A cromatografia é uma técnica usada para separação e/ou purificação de diferentes biomoléculas com finalidade analítica ou preparativa. Esta técnica tem sido muito aplicada na área da indústria farmacêutica, para a obtenção de proteínas e ácidos nucleicos, entre outras biomoléculas. Com o passar dos anos, a quantidade de amostras e a pureza necessária aumentaram, tornando esta técnica numa das mais usadas na indústria biotecnológica. O desenvolvimento de novas matrizes cromatográficas tem sido um tema de grande importância, a fim de encontrar uma matriz ideal que reúna características como custos associados, eficiente, estável física e quimicamente, com uma elevada eficiência de transferência de massa e reutilizável. A gelana é um polímero polissacárido natural linear que tem sido aplicado em diversas áreas, como indústria alimentar (agente gelificante e espessante), indústria farmacêutica (entrega direcionada de fármacos), podendo também ser aplicada como um substituinte do agar. Este polímero linear em determinadas condições pode sofrer uma alteração conformacional, formando uma dupla hélice. Na presença de catiões, forma uma rede tridimensional, devido às interações ocorridas entre a gelana, os catiões e as moléculas de água em solução, dando origem a um gel. Tendo em conta estas considerações, o presente trabalho foi realizado com o intuito de formular um gel de gelana estável, a fim de adquirir um comportamento de matriz cromatográfica. A preparação dos géis foi feita de acordo com os seguintes parâmetros: concentração de gelana, concentração de catião (zinco), concentração de DMF, temperatura e tempo de reação. Uma vez que a estabilidade dos géis de gelana é afetada por todos estes parâmetros, foram preparadas várias formulações em que se variou apenas um destes parâmetros para verificar qual o seu efeito na estabilidade do gel. As formulações foram testadas de acordo com as seguintes variações: concentração de gelana (0,75% - 2%), concentração de sulfato de zinco (30 – 120 mM), concentração de DMF (0% - 30%), temperatura (temperatura ambiente – 110ºC) e tempo de reação (0.5 horas – “overnight”). Posteriormente foi aplicada uma estratégia de desenho experimental no sentido de definir as condições ideais para a preparação do gel, e consequentemente obter um gel mais estável. Assim, os melhores resultados quanto à estabilidade do gel foram obtidos quando se usou 0,75% de gelana, 48 mM de sulfato de zinco, 0 % DMF, 25ºC, e 0,5 horas. Adicionalmente e aproveitando a natureza aniónica do polímero de gelana, foi possível explorar diferentes interações com as seguintes proteínas modelo (albumina sérica bovina, αquimotripsina e lisozima). Na presença do tampão MÊS com pH 6,2, a albumina sérica bovina encontrava-se com carga negativa, enquanto que a α-quimotripsina e lisozima se encontravam com carga positiva, tendo em conta os respetivos pontos isoelétricos. Sendo assim, a albumina sérica bovina não interagiu com a matriz, visto que ambas têm carga negativa. Pelo contrário, a α-quimotripsina e lisozima ligaram-se à matriz, devido à oposição de cargas. A separação das três proteínas foi conseguida através da eluição por passos, com o aumento gradual da concentração de NaCl no tampão de eluição. A albumina sérica bovina foi a primeira proteína a eluir após a aplicação da amostra, ainda com a passagem do tampão de eluição sem sal. Posteriormente aumentou-se a concentração de sal promovendo-se a eluição da α-quimotripsina . Por fim, para concentrações de sal mais elevadas, e a lisozima que promoveu uma interação mais forte com a matriz de gelana acabou por eluir também. A fim de melhor caracterizar esta nova matriz, foi determinada também a capacidade dinâmica de ligação utilizando uma estratégia de saturação da coluna com uma solução de lisozima 0,05 mg/mL. Os valores obtidos para a capacidade dinâmica de ligação da gelana a 10% e a 50% foram 3,9 mg/ml e 17,4 mg/ml, respetivamente. Comparando com outras matrizes cromatográficas, os valores de capacidade dinâmica de ligação da matriz de gelana estão dentro do esperado. Estes estudos iniciais permitiram concluir que a gelana interagiu com as proteínas, considerando a diferença de cargas, e que a sua eluição também foi possível com o aumento de sal, o que revela uma provável potencialidade como matriz cromatográfica de troca catiónica. Para poder afirmar e provar que o polímero gelana pode constituir uma matriz cromatográfica, devem ser feitos mais ensaios. Nomeadamente testar a purificação de amostras mais complexas e determinar mais parâmetros (como por exemplo a capacidade iónica e o tipo e tamanho de poros) que permitam a otimização da matriz. No futuro, poderemos vir a ter uma matriz versátil com aplicação em diversos domínios científicos.

Chromatography is one of the most studied methods, due to its simplicity, versatility and high reproducibility, to separate and purify molecules that can have therapeutic, industrial and biotechnological interest. In recent years, the development of new chromatographic matrices has been continuously increased in order to afford rapid and efficient separations and decrease the use of resources. Gellan gum is a natural anionic exopolysaccharide and, in the presence of divalent cations, has the ability to form thermos-reversible strong gels resistant to temperature and extreme acidic conditions. In this work, it was proposed the preparation of gellan gum microbeads to be used as a stable chromatographic stationary phase. In order to produce the matrix, a low-cost water-in-oil emulsion technique was adopted. To obtain optimal conditions to the bead formulation, experimental design was applied, which allowed the optimization of the experimental conditions and to produce microbeads with the smallest diameter possible. Due to the negative charge of gellan gum, it was possible to study the interactions established with three model proteins (BSA, a-chymotrypsin and lysozyme) and with a therapeutic complex protein, SCOMT. In the model protein assays, MES buffer with pH 6.2 was used, which conferred negative charge to BSA and positive charge to a-chymotrypsin and lysozyme, due to its isoelectric points. Thus, BSA did not bind to the matrix while the other two proteins were retained to the gellan gum, being eluted with an increase of ionic strength. Regarding to prepurified SCOMT sample, a buffer with pH 4.0 was used under equilibrium conditions, conferring positive charge to the protein, thus, it also interacted with the column and was majorly eluted by pH manipulation (by changing the buffer pH to 6.4), allowing the elimination of some protein contaminants. Dynamic binding capacity assay of the gellan gum microbeads was made, in order to characterize this support as a novel chromatographic matrix. The values of DBC of the gellan gum stationary phase to 10 % and 50 % of breakthrough were 2.43 mg/mL and 4.73 mg/mL, respectively. These DBC values are satisfactory when compared to commercial resins used in affinity chromatography, taking into account that protein interaction only occurred at the gellan bead surface. These results indicated that gellan gum microbeads obtained by waterin- oil emulsion technique can be used as an innovative and promising chromatographic support due to its gelling ability and versatility to interact with different biomolecules.
A cromatografia é um dos métodos mais usados para a separação e purificação de diferentes biomoléculas terapêuticas. É uma técnica que tem sido muito explorada, ao longo dos últimos anos, nas áreas da indústria farmacêutica e biotecnológica para a obtenção de proteínas e ácidos nucleicos com elevado grau de pureza. Os suportes cromatográficos têm sido alvo de grandes desenvolvimentos a fim de encontrar uma matriz que reúna as características ideais como porosidade, estabilidade química e física, elevada eficiência de transferência de massa, baixo custo de produção associado, biocompatibilidade, hidrofilicidade, boa capacidade de manutenção do fluxo e capacidade de reutilização após vários ensaios cromatográficos. A goma de gelana é um polímero polissacárido natural linear que tem uma vasta gama de aplicações em diversas áreas, desde a indústria alimentar (agente espessante e gelificante), cosmética (loções, cremes, maquilhagem, etc), indústria biotecnológica (como um substituinte do agar), indústria farmacêutica (sistema de entrega direcionada de fármacos e microencapsulações) e medicina (construção de “scaffolds” para regeneração de tecidos). Devido às suas propriedades como versatilidade, biocompatibilidade, biodegradabilidade e elevada estabilidade, a gelana tem atraído grande interesse por parte do mercado e tem ganho crescente importância entre as diferentes indústrias. A gelana possui a capacidade de, em determinadas condições e na presença de catiões divalentes, sofrer uma alteração conformacional formando uma forte rede tridimensional devido às interações entre a gelana, os iões e as moléculas de água dando origem a um gel termoreversível, resistente a altas temperaturas e a extremas condições ácidas. Tendo em conta as características apresentadas, o presente trabalho teve como objetivo otimizar a formulação de uma matriz cromatográfica pela técnica de emulsão água-em-óleo a fim de ser usada em diversos processos cromatográficos. A inovação deste projeto tem como base a produção de microesferas através de um método de emulsão água-em-óleo, usando dois líquidos imiscíveis, a água e um óleo alimentar vegetal. Trata-se de uma técnica que não requer o uso de instrumentos complexos, é de fácil manuseamento, tem baixo custo associado e permite o fácil controlo dos parâmetros associados a este processo. A preparação das esferas foi feita de acordo com os seguintes parâmetros: concentração da goma de gelana (1 % - 2,5 %), velocidade de agitação (250 rpm – 750 rpm) e temperatura (20 ºC – 100 ºC). Considerando que todos estes fatores afetam a estabilidade e estrutura das microesferas, foram feitas várias formulações utilizando um desenho experimental, estratégia que permitiu obter as condições ótimas no sentido de se produzirem micropartículas com o mínimo diâmetro possível. As condições ótimas fornecidas pelo desenho foram 1,41 % de concentração de gelana, 749,47 rpm de velocidade de agitação e 99,20 ºC para obter microesferas com um tamanho de 277,08 µm. Para a formulação deste suporte cromatográfico, foi necessário a presença de catiões divalentes numa solução de reforço, para onde as esferas foram transferidas a fim de aumentar a estabilidade. O catião escolhido foi o bário, que devido ao seu grande tamanho iónico é considerado bastante eficaz na gelificação da gelana, pois contribui largamente para a formação da estável e rígida rede tridimensional. Além disso, o bário também permitiu a formação de esferas uniformes de tamanho reduzido com um pequeno ratio de inchaço. Para a validação do modelo fornecido pelo software do desenho experimental, foi necessário efetuar cinco réplicas de dois pontos previstos dados pelo programa. A visualização das esferas produzidas foi feita através de microscopia ótica que permitiu a medição dos diâmetros das micropartículas e através de microscopia eletrónica de varrimento, que tornou possível a análise estrutural das esferas num estado liofilizado, bem como a análise topográfica e morfológica da sua superfície. Além disso, esta última metodologia também permitiu avaliar a porosidade das esferas, de onde foi constatado a ausência de canais interiores, sugerindo que qualquer interação estabelecida com a matriz de goma de gelana se dê apenas na sua superfície. Aproveitando a natureza aniónica do polímero de gelana e o facto de não ter que ser funcionalizada com ligandos como os suportes cromatográficos comerciais, foi possível explorar diferentes interações com as proteínas modelo albumina sérica bovina (BSA), a-quimotripsina e lisozima, bem como com um extracto parcialmente purificado da proteína catecol-O-metiltransferase humana na isoforma solúvel (hSCOMT). Nos ensaios com as proteínas modelo, o tampão usado foi o MES a pH 6.2, o que conferiu carga negativa à BSA e cargas positivas à a-quimotripsina e lisozima devido aos seus pontos isoelétricos. Assim, a BSA não ligou à matriz tendo sido eluída após a injeção da amostra na coluna, ainda com a passagem do tampão sem sal, enquanto as outras duas proteínas interagiram devido à oposição de cargas, tendo sido eluídas com o aumento da força iónica. Quanto à amostra pré-purificada de SCOMT, foi usado um tampão de pH 4.0 nas condições de equilíbrio, o que conferiu carga positiva a esta proteína. Esta condição permitiu a interação desta proteina com a coluna, tendo sido maioritariamente eluída através da manipulação do pH (alterando o pH do tampão para 6.4), permitindo assim a eliminação de algumas proteínas contaminantes presentes na amostra. A fim de se caracterizar melhor esta nova matriz cromatográfica, foi determinada a capacidade dinâmica de ligação utilizando uma estratégia de saturação da coluna com uma solução de lisozima 0,5 mg/mL a 1 mL/min. Os valores obtidos na capacidade de ligação das microesferas de goma de gelana a 10 % e 50 % da curva de saturação foram 2,43 mg/mL e 4,73 mg/mL, respetivamente. Comparando com outras matrizes cromatográficas comerciais e tendo em conta que apenas a área de superfície das microesferas de gelana é funcional, estes valores estão dentro do esperado. Estes estudos permitiram concluir que a estabilidade da matriz cromatográfica de gelana foi incrementada com o desenvolvimento deste projecto de mestrado e que permitiu a interação com proteínas de várias naturezas, através de estratégias de eluição com manipulação de força iónica e pH. Em suma, os dados apresentados manifestam uma versatilidade da gelana em interagir com diferentes biomoléculas e, devido à sua capacidade de gelificação, foi possível a elaboração deste inovador e promissor suporte cromatográfico para a cromatografia de troca catiónica, a partir de microesferas da goma de gelana.

碩士
中山醫學大學
醫學檢驗暨生物技術學系碩士班
102
About oral cancer pathogenesis, more research is focused on the chemical factor induced cell proliferation and progression, however, physical stimulation may also induce reasons. Relations mechanical stimulation and cancer in breast cancer, oral cancer aspects have wanted more research. In the past, construction of 3D culture system of cancer cells in vitro using Matrigel as a substrate, this study is based on microbial polysaccharides Gellan gum hydrogel to construct 3D cell culture system. Gellan gum form gels with divalent cations, preparation of 0.6% and 0.8% of Gellan gum with 0.5% CaCl2 to become a 3D structure of the gel, and co-cultured with the oral cancer SCC15 (squamous cell carcinoma 15) to form tumor sphere. By WST-1 test preliminary experimental results are shown in 3D and 2D culture 24 hrs, 48 hrs and 72 hrs after, materials for oral cancer SCC15 has no significant differences. In addition, the 3D hydrol gel was also used as a fat-soluble drugs and water-soluble drugs of drug transport carrier. Confirmed by the findings, Gellan gum gum as water soluble drug transport vector, 3D structures will disintegrate in 48 hours; Encapsulating of a lipophilic drug, Gellan gum structure can be maintained over two weeks. The research results will be applied in the future of the in vitro research platform and oral cancer treatment.

碩士
中山醫學大學
醫學檢驗暨生物技術學系碩士班
102
In this study we prepared thick films of Gellan gum (GG)/ Graphene (GO) for wound-dressing applications. Gellan gum, an anion polysaccharide, it has biocompatibility and low production cost. Graphene, a single-atom-thick of carbon atoms, it possesses thermal, mechanical, and electrical properties. The applied film was fabricated using two different methods. The first method used of gellan gum mixed with graphene and reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to obtain a cross-linked film (GGM). The second method that of the prepared graphene is spray on gellan gum film, and then through EDC cross-linking to form the GGS film. The GGM with 63.97% gel content and 94.27% water content. The GGS with 60.82% gel content and 95.72% water content. Antimicrobial susceptibility test has resulted that GGM and GGS without effective antimicrobials for prevention and treatment of infections. Animal wound healing study, on day3, 7, 14, after surgery, the wound size reduction (%) in the GGM-treated group was 25.85%、45.18%、88.98%. On day3, 7, 14, after surgery, the wound size reduction (%) in the GGS-treated group was 20.85%、36.88%、75.38%. Thus, the GGM, GGS films developed in this study has potential for future use in surgical applications.

Tese de Doutoramento em Ciências da Saúde
Spinal Cord Injury (SCI) is a highly debilitating condition, with devastating consequences for the patients and their families. Partial or complete loss of motor and sensorial functions below the level of injury are the main visible outcomes. However, cardiac and respiratory compromise, together with bladder and bowel dysfunctions, among others, are frequently observed, impairing significantly the quality of life. Currently there are no treatments for SCI, with most approaches being used in order to minimize injury progression. The lack of treatments can be attributed to the aggressive pathophysiology of SCI. The initial impact immediately leads to massive tissue damage and neuronal death, with edema formation and hemorrhages. For the next minutes up to several weeks, a cascade of cellular and molecular secondary alterations is triggered, leading to further neuronal damage. Finally, in the chronic phase, a glial scar and a cystic cavity are formed, creating a physical and chemical barrier to regeneration. Considering the injury complexity and the multitude of secondary alterations that can be targeted, it is more likely that a combinatorial therapeutic approach will have more impact than single-based therapies. Among the different approaches, tissue engineering (TE) and regenerative medicine concepts could be of extreme relevance for SCI. Cellular transplantation, one of the cornerstones of TE, has been applied in SCI research for decades. Nonetheless, and despite the numerous studies in the field, most of them fail when translated into the clinics. Combining biomaterial-based strategies with cell therapies could solve some of the associated problems, such as the low cell survival after transplants. In this way, the work developed during this thesis aimed to develop a combinatorial approach, conjugating the transplantation of two different cellular populations with a functional hydrogel, serving both as a physical matrix for neuronal regrowth and as a vehicle for cell transplantation. Thus, gellan gum hydrogel, previously modified with fibronectin-derived peptides (GGGRGDS), was combined with adipose tissue-derived stem/stromal cells (ASCs) and olfactory ensheathing cells (OECs). Both cell types have been associated with promising protective and regenerative effects, in different models of SCI. In this work, we firstly showed that ASCs and OECs could grow in vitro, in direct coculture systems, without affecting their morphology, growth and viability. In addition, their combination was beneficial in vitro, resulting in increased axonal growth using a model of axonal regeneration (Dorsal Root Ganglia – DRG – explants). Then, ASCs/OECs were applied as a single transplantation into a rat thoracic hemisection model of injury (T10 level). The treatment led to significant recovery of hindlimb function, which was associated with reduced levels of inflammation at the lesion site. Later, cells were encapsulated within GG-GRGDS hydrogels and demonstrated a normal morphology and growth in vitro. Moreover, their combined application into a lumbar hemisection model, led to a significant recovery of locomotor function. The histological analysis showed reduction in inflammation, astrogliosis and an increased number of neurofilament positive staining, possible indicative of axonal regeneration or neuronal protection. Finally, the same strategy was implemented in a cervical model of injury, which induces respiratory deficits, affecting diaphragmatic function. Following a C2 hemisection, hydrogel and cells were transplanted at the injury site, immediately after lesion. The combinatorial treatment induced the most significant recovery of diaphragmatic function, as assessed by electromyogram activity of the ipsilateral hemi-diaphragm. Both ventral and medial portions of the diaphragm were partially recovered. Moreover, and despite no differences were seen in forelimb motor function, GG-GRGDS and ASCs/OECs induced a significant recovery of sensorial perception, in the contralateral forelimb, as measured by the Von Frey test. All together, these results disclose a great therapeutic potential for the application of GGGRGDS hydrogels with ASCs and OECs, since its application to different models of injury resulted in significant and relevant improvements of function. Not only motor behaviors associated with locomotion were recovered, but also motor control of the diaphragm, essential for respiration, and also sensorial perception recovery. In addition, all components of this approach could be translated to the clinics, as ASCs and OECs can be obtained from autologous sources and gellan gum proved to be biocompatible, with low immunogenic potential. This work presents an important strategy for SCI repair, that with further pre-clinical testing in larger animal models, could be one day applied to the clinics.
As lesões vertebro-medulares (LVM) são uma condição debilitante, com consequências devastadoras quer para pacientes como para as suas famílias. A perda parcial ou total das capacidades motoras e sensoriais abaixo do local da lesão é a consequência mais notória. Contudo, problemas cardíacos e respiratórios, assim como disfunção dos sistemas urinário e gastrointestinal, entre outros, acontecem frequentemente, reduzindo significativamente a qualidade de vida. Atualmente não existem tratamentos para as LVM, com a maioria das abordagens a ser utilizada apenas de modo a minimizar a progressão da lesão. A falta de tratamentos pode ser atribuída à agressiva fisiopatologia da doença. O impacto inicial leva a um dano tecidular e morte neuronal imediatas, com formação de edema e hemorragia. Durante os minutos seguintes, até várias semanas, uma série de alterações celulares e moleculares é ativada, conduzindo a um aumento do dano neuronal. Por fim, na fase crónica, são formadas a cicatriz glial e a cavidade cística, criando uma barreira física e química à regeneração. Tendo em conta a complexidade e a panóplia de alterações secundárias que podem ser usadas como alvo terapêutico, é provável que tratamentos baseados em abordagens combinatórias terão um maior impacto do que estratégias singulares. Dentro das várias possibilidades, os conceitos usados em engenharia de tecidos e medicina regenerativa poderão ser de uma grande relevância para as LVM. O transplante celular é um dos pilares da engenharia de tecidos e tem vindo a ser aplicado na investigação em LVM desde há décadas. Não obstante, e apesar do elevado número de trabalhos neste campo, a grande maioria falha quando tenta ser aplicado na clínica. Combinar estratégias baseadas em biomateriais com terapias celulares poderá resolver alguns dos problemas associados a esta abordagem, nomeadamente a baixa sobrevivência das células após transplante. Deste modo, o trabalho desenvolvido durante esta tese teve como objetivo desenhar uma abordagem combinatória, conjugando o transplante de duas populações celulares distintas com um hidrogel modificado, que serve tanto como uma matriz física para o crescimento neuronal, assim como um veículo para o transplante celular. Neste sentido, o hidrogel goma gelana, previamente modificado com péptidos derivados de fibronectina (GG-GRGDS), foi combinado com células estaminais do tecido adiposo (ASCs) e células envolventes do bolbo olfativo (OECs). Ambos os tipos celulares têm sido associados a efeitos neuro-protetores e neuro-regenerativos, em diferentes modelos de LVM. Neste trabalho, primeiramente demonstramos que ASCs e OECs crescem in vitro, em sistemas de co-cultura direta, sem que sejam afetadas as suas morfologias, proliferação e viabilidade. Para além disso, a sua combinação foi benéfica, levando a um crescimento de neurites, num modelo in vitro de regeneração axonal. De seguida, ASCs/OECs foram transplantadas para um modelo de rato de hemisecção torácica (nível T10). O tratamento induziu uma recuperação significativa da função das patas traseiras, o que esteve associado a níveis reduzidos de inflamação no local da lesão. Mais tarde, as células foram também encapsuladas em hidrogéis de GG-GRGDS e apresentaram uma morfologia e crescimento normais in vitro. Adicionalmente, a sua aplicação conjunta num modelo de hemisecção lombar (mais agressivo) levou a uma melhoria significativa da locomoção. A análise histológica permitiu perceber uma redução da inflamação, da astrogliose e aumento da marcação para neurofilamento, possível indicador de regeneração axonal ou de maior proteção neuronal. Por fim, a mesma estratégia foi implementada num modelo de lesão cervical, que induz défices respiratórios, afetando a função do diafragma. Após hemisecção em C2, hidrogel e células foram imediatamente transplantados, no local de lesão. O tratamento combinado levou a uma maior recuperação da função do diafragma, avaliada por eletromiograma do músculo ipsilateral do diafragma. As regiões mais dorsal e medial do diafragma demonstraram uma recuperação parcial da função. Apesar de não terem sido observadas diferenças na função motora das patas dianteiras, a terapia combinatória ajudou a recuperar a função sensorial, da pata dianteira contralateral, após avaliação com filamentos de Von Frey. Resumindo, estes resultados revelam um grande potencial terapêutico para o uso de hidrogéis de GG-GRGDS com ASCs e OECs, dada a sua aplicação com sucesso a diferentes modelos de lesão, com melhorias de função significativas. Não só comportamentos como a locomoção foram melhorados, mas também o controlo do diafragma foi recuperado parcialmente, o que é essencial para a função respiratória, e ainda a perceção sensorial. Mais ainda, todas as componentes desta abordagem podem ser facilmente aplicadas na clínica, visto ser possível obter ASCs e OECs de uma maneira autóloga (do próprio paciente) e ao facto da goma gelana ser biocompatível, com baixa probabilidade de provocar uma reação imune. Este trabalho apresenta uma abordagem relevante para as LVM, que após mais estudos pré-clínicos em animais de maior porte, poderá um dia vir a ser aplicado em pacientes.
The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), athe Schol of Medicine, University of Minho. Financial suport was provided by Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-17-2013 and MC-04-2017); Portuguese Foundation for Science and Technology (Doctoral felowship to Eduardo Gomes SFRH/BD/103075/2014, Post-doctoral to Nuno Silva BPD/9701/2013, IF Development Granto A. J. Salgado). This thesis was performed under the Memorandum of understanding on Academic exchanges betwen Schol of Medicine, University of Minho and the Luso-American Development Foundation. This thesis was also funded by national funds through FCT under the scope grant reference TUBITAK/07/2014 and 359-PCDT Project: PTDC/DTP-FTO/5109/2014. Suport was also provided by: Craig H. Neilsen Foundation (grant #47686 to A.C. Lepore) and the National Institute of Neurological Disorders and Stroke (#2R01NS079702; 1R01NS10385; #1R56NS096028 to A.C. Lepore). This thesis has been developed under the scope of the projects NORTE-01-0145-FEDER-013, suported by Northern Portugal Regional Operational Programe (NORTE 2020), under the 2020 Partnership Agrement, through the European Development Fund (FEDER). This work has ben funded by FEDER funds, through the Competitivenes Factors Operational Programe (COMPETE), and by National funds, through the Foundation for Science Technology (FCT), under scope of the project POCI-01-0145- FEDER-07038. ASCs were kindly provided by Prof. Jef Gimble (LaCel Inc, USA).

碩士
國立臺南藝術大學
博物館學與古物維護研究所
103
For decades, scientists and conservators have been working on researching and finding appropriate treatments of the iron-gall ink archives. There are a lot of iron-gall ink archives and they are very important and precious. This made finding a solution to the conservation and treatments of iron-gall ink archives all the more important. Hence, the goal of this paper is to find a way to remove iron-gall ink archives’ backing without affecting the soluble Fe (II) ions. To do so, this paper looks at various worldwide researches to understand the properties, making process, degradation factors and treatments ofiron-gall ink, and also the properties of Gellan gum and its use on conservation. Experiments are done using two methods - rigid Gellan gums and brushing solvents, to remove iron-gall ink archives’ backing and test the soluble Fe (II) ions with bathophenanthroline/ethanol solvent. By experimenting with three different kinds of rigid Gellan gums and three different solvents with different humidified levels, the best results were found - when one uses 3% Gellan gum/calcium phytate and brushes by 25:75 calcium phytate/ethanol solvent. In conclusion, the results of brushing calcium phytate/ethanol solvent is good, but we also have to make sure the iron-gall ink is insoluble. Also, before using Gellan gums, we have to consider the water content and penetration speed, which could be adjusted by the percentage and thickness of Gellan Gum. We could also add cation into Gellan gum to increase its intensity. The most important is the control of the environment for storing these archives. If under appropriate relative humidity, lighting, temperature, anoxic and alkaline conditions, the damage to the archives may be reduced and they may be preserved longer.

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais
Regeneration, reconstruction and repair of peripheral nerve injuries (PNIs) are among the most complex and demanding challenges in the field of regenerative medicine. As a promising alternative to the “gold standard” autologous nerve grafts, tissue-engineered nerve guidance conduits (NGCs) have been extensively studied. However, in order to be able to produce an adequate NGC, the basic principles of neuro-biology must be known and followed. Also, great efforts have been made in terms of pre-clinical and clinical applications of engineered biomaterials for peripheral nerve regeneration. Furthermore, nanotechnology approaches have propelled nerve regenerative strategies. These themes are the focus of Section 1, in Chapters I, II and III. This PhD project has focused mainly on researching, developing and testing natural and biodegradable biomaterials using several fabrication methods to obtain the NGCs (Chapter IV). The first explored biomaterial was chitosan, in Chapter V, where the suitability of chitosan membranes with three different low degrees of acetylation was evaluated. Furthermore, to enhance the membranes’ cell adhesion and angiogenic properties, extracted human hair keratin was combined to the previous described membranes (Chapter VI). In Chapter VII, chitosan NGCs which are currently being used in in the clinical setting (Reaxon®), were used in combination with different gellan gum luminal fillers, mimicking the native Bands of Büngner. In Chapter VIII, a ground-breaking silk fibroin (SF) fabrication method for NGCs was established. SF NGCs were produced using a novel technology that comprises a horseradish peroxidase-mediated crosslinking system. Such innovative processing method resulted in the filling of an international patent. The excellent outcomes demonstrated by the enzymaticallycrosslinked method lead to an original research paper (Chapter IX). In line with the previous work, and because of its resourcefulness, a step forward was given, and a state-of-the-art study was performed, comprising the production of enzymatically-crosslinked SF NGCs capable of incorporating and delivering different neurotrophic factors. The developed NGCs restored the functional activity and enhanced neovascularization in the regenerated nerves, revealing comparable results to the autograft (Chapter X). In summary, the presented outcomes contributed to improve the actual state-of-art in what regards peripheral nerve regeneration, providing promising and versatile alternatives by using different types of natural-based biomaterials and approaches (Chapter XI).
A regeneração, reconstrução e reparação das lesões do nervo periférico encontram-se entre os desafios mais exigentes e complexos na área da Medicina Regenerativa. Como tratamento alternativo aos enxertos de nervo autólogo, Estruturas tubulares e protetoras do nervo produzidos, através de técnicas de Engenharia de Tecidos têm sido extensivamente estudados. Para além disso, grandes esforços têm sido feitos em termos de aplicações clínicas e pré-clínicas de biomateriais para a regeneração do nervo periférico. Abordagens nanotecnológicas têm também impulsionado o avanço nesta área. Estes temas são abordados na Secção 1, nos Capítulos I, II e III. Este projeto de Doutoramento focou-se principalmente na investigação, desenvolvimento e testes laboratoriais de várias formulações de biomateriais, combinados com diferentes técnicas de fabrico (Capítulo IV) de estruturas tubulares, usando apenas polímeros biodegradáveis conjugados com outras funcionalizações físicas ou biológicas. O quitosano foi o primeiro material investigado, no Capítulo V e VI, onde foram estudadas diferentes membranas de quitosano, assim como a melhoria das propriedades biológicas e angiogénicas destas, através da adição de queratina extraída de cabelo humano. No Capítulo VII, tubos de quitosano atualmente usados em Humanos (Reaxon®) foram combinados com diferentes formulações de Gellan Gum, produzidos para preencher o lúmen dos tubos, mimetizando assim as bandas de Bungner. No Capítulo VIII, um inovador método de produção de tubos condutores à base da Fibroína da Seda foi desenvolvido, resultando no registo de uma patente internacional. Esta metodologia envolve o uso de um sistema de reticulação enzimática com base na enzima peroxidase e o peróxido de hidrogénio (oxidante). Os excelentes resultados demonstrados através deste sistema de produção de tubos originaram um artigo original (Capítulo IX). Seguindo a mesma linha de trabalho, e devido á versatilidade da técnica, estudos inovadores foram desenvolvidos, onde fatores neurotróficos foram incorporados nos tubos de Fibroína da Seda, com o objetivo de restaurar a funcionalidade motora assim como a re-vascularização dos nervos regenerados (Capítulo X). Em suma, os resultados apresentados nesta tese indicam a versatilidade dos diferentes biomateriais e formulações testadas, assim como das diferentes técnicas, contribuindo deste modo para melhorar o atual estado da arte no que diz respeito à regeneração do nervo periférico (Capítulo XI).

The water sorption of the films of polyacrylic acid, guar gum, gellan gum and PAA-guar, PAA-gellan mixtures of different compositions, prepared at 25°C and 70°C, was studied. The chemical potentials of water Δμ1, polymer component Δμ2, the average Gibbs energy of interaction of polymers and mixtures with water Δgm, the average Gibbs mixing energy of PAA and guar, PAA and gellan Δgx have been determined. It is established that the increase in the preparation temperature significantly enhances the films’ sorption capacity in relation to water. It is determined that the Gibbs mixing energy of polyacrylic acid with guar and gellan are negative for all component ratios (Δgx <0). The second derivative of Gibbs energy for PAA-guar mixtures has different signs, and in a wide range of concentrations of the mixture it is negative. This indicates the absence of compatibility and the formation of two-phase colloidal systems in mixtures of guar with PAA. In contrast to this for PAA-guar mixtures the second derivative of Gibbs energy is positive for all component ratios. This indicates the polymer compatibilityю The increase in the preparation temperature of mixtures leads to an increase in the interaction of the components in the two-phase system, which may be related to the reduction in size of the associates of macromolecules of guar in aqueous solutions.
Изучена равновесная сорбция паров воды пленками полиакриловой кислоты ПАК, гуара геллана и смесями ПАК-гуар разного состава, приготовленными при 25 °C и 70 °C. Рассчитаны разности химических потенциалов воды Δμ1, полимерных компонентов Δμ2, средние удельные энергии Гиббса взаимодействия полимеров и смесей с водой Δgm и друг сдругом Δgx. Установлено, что повышение температуры приготовления пленок значительно увеличивает их сорбционную способность по отношению к воде. Обнаружено, что энергии Гиббса смешения гуара и геллана с полиакриловой кислотой отрицательны при всех соотношениях компонентов (Δgx < 0). Вторая производная энергии Гиббса по составу имеет разные знаки, и для системы гуар – ПАК в широкой области концентраций смеси она отрицательна. Это свидетельствует об отсутствии совместимости и образовании двухфазных коллоидных систем в смесях гуара с ПАК. В противоположность этому для системы ПАК-геллан величина положительна во всей области составов. Это свидетельствует о совместимости полимеров. При этом повышение температуры приготовления смесей приводит к усилению взаимодействия компонентов в двухфазной системе, что может быть связано с уменьшением размеров ассоциатов макромолекул гуара в водных растворах при повышении температуры.

Selected food gums (hydrocolloids) were tested for their abilities to encapsulate enzymes for acclerating cheese ripening. The effect of pH and acidity on gel strength of the gums was determined. Enzyme was entrapped in k-carrageenan, gellan gum and milk fat and incorporated into cheese milk prior to cheese making. The cheese produced was tested for protein breakdown and amino acid production during ripening and textural and sensory properties of the ripened cheese were also evaluated. The findings and significance of this study and the literature review are presented. Gels were produced from alginate. Most gels showed reduced strength after treatment in solutions of either modified acidities or pH. There was, however, no significant change in gel strength between the different treatments for most of the gums. The activity of encapsulated enzyme were also investigated. Enzymes encapsulated in k-carrageenan, gellan and alginate gums retained higher activities than the other gums studied. Enzymes entrapped in agar gels had the least retention of activity. The retention of enzyme capsules produced from gellan, k-carrageenan and milk fat in cheese curd was investigated. Loss of encapsulated enzymes in cheese whey was also determined. Enzyme loss in the whey ranged from 5.6 to 17.9% with the highest losses observed with milk fat and the least with gellan gum capsules. Most of the cheeses treated with enzyme capsules showed higher levels of amino acid within two weeks than control cheese. After two weeks, all experimental cheeses showed higher production of amino acids than the control cheese. The addition of enzyme capsule to cheese did lead to a higher growth level of microorganisms. The experimental cheese exhibited lower score than the control cheese for most textural properties. The experimental cheeses were not significantly different in flavour and aroma from the control cheese. K-carrageenan treated cheeses were recorded as having the highest score for bitter after taste. Except for those cheeses treated with k-carrageenan capsules, the overall acceptability for the trial cheeses were not significantly different from that of 6 month old untreated cheese.

Tese de Doutoramento - Programa Doutoral em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais
The incidence of Diabetic Foot Ulcerations (DFUs) is increasing with the emergent worldwide prevalence of Diabetes. DFUs pathophysiologies such as delayed and immature re-epithelialization, persistent inflammation, ischemia and neuropathy, hinder the healing of wounds which rapidly develop into DFUs. The main problematic of this chronic and overwhelming condition is the time of healing that may last months or lead to foot amputation due to non-healing. Hence, there is an urgent need to find an effective treatment for diabetic wounds. The remarkable capacity of hydrogels to absorb and retain water has been recognized as highly valuable in providing and maintaining wound moist. Furthermore, it is currently demonstrated that hydrogels per se, or carrying different bioactive agents and/or cells, are bioresponsive. In this thesis, we hypothesized that both naturally bioactive polymers and stem cell-carrying hydrogels would be suitable to target the characteristic DFUs pathophysiologies. Gellan gum (GG) hydrogels have been extensively studied for different biomedical applications. However, the reduced resilience to deformation, the lack of intrinsic cell-adhesive features and cellmediated degradation are not ideal for skin wound healing. The first aim of this thesis was to overcome these limitations. The absence of cell adhesion moieties was surpassed through two different strategies: microstructural rearrangement of GG hydrogels generating GG-based spongy-like hydrogels, and chemical addition of peptide cell adhesive sequences to GG previously modified with divinyl sulfone (GGDVS). Moreover, cell-mediated degradation was attained by intercalating a cell-degradable polymer, hyaluronic acid (HA), within the core of spongy-like hydrogels and using a dithiol crosslinker sensitive to metalloproteases. Overall, both spongy-like hydrogels and peptide-modified GGDVS hydrogels showed ameliorated mechanical performance that was also tuned by varying the crosslinker and polymer amount. The importance of creating two different GG-based matrices with hydrogels characteristic features relies on their individual properties that can be advantageous for treating skin wounds. Dried polymeric networks, before being hydrated to form the spongy-like hydrogels, can be stored off-theshelf and produced in any form or shape-fit according to the wound size. These low-cost and ready-touse dried materials have great ability to absorb the wound exudate. Moreover, dried polymeric networks can be re-hydrated with any water-based solution with/without cells or bioactive agents immediately before application into the wound. In addition to the ability of carrying cells or bioactive agents, GGDVS hydrogels have the advantage of being injectable, which might not be immediately recognized as relevant for skin applications but can be of great importance for the treatment of interdigital wounds. The capacity of spongy-like hydrogels to directly influence keratinocytes was addressed after the incorporation of Sepia officinalis eumelanin to attain a system of eumelanin sustained release. The ability of primary human keratinocytes to phagocyte Sepia officinalis eumelanin, which was then accumulated in cytosolic vesicles and nuclei surroundings, was demonstrated. Keratinocytes viability and maturation was not affected by the contact with eumelanin but cell morphology and cell proliferation was dependent on eumelanin concentration. A time- and eumelanin concentrationdependent reduction of the amount of ROS/RNS released by UVA/UVB irradiated hKCs pre-exposed to it was observed. Hence, the effects of eumelanin over keratinocytes can be advantageous to benefit skin healing. The possibility to confer an angiogenic intrinsic character to the GG matrices was investigated following two approaches, the entanglement of HA within GG polymeric networks (GG-HA) and the modification of GGDVS with T1 or C16 peptides, from CCN1 and laminin proteins, recognized by endothelial cells. The incorporation of HA within GG polymeric networks resulted in spongy-like hydrogels degradation by hyaluronidase and consequent formation of low molecular weight HA fragments capable to improve blood perfusion and neovascularization in mice ischemic hind limbs. Although the angiogenic potential of T1 and C16 peptides was not studied, endothelial cell adherence and spreading within GGDVS hydrogels modified with T1 or C16 peptides was dependent on the type and amount of peptide. Cells presented a cobblestone-like morphology within GGDVS hydrogels modified with the C16 peptide, while a polarized morphology was observed with the T1 peptide. Motivated by our previous results, a strategy that concomitantly modulates inflammation and angiogenesis, and promotes neoinnervation, was followed. Aiming to potentiate a secretome relevant for nerve regeneration, human adipose-derived stem cells (hASCs) were pre-cultured in GG-HA spongylike hydrogels under neurogenic conditioned or standard media prior transplantation into full-thickness skin wounds of diabetic mice. Faster wound closure and re-epithelialization was attained 4 weeks posttransplantation for the majority of the wounds, but a thicker and fully matured epidermis was observed for the stem cell-laden GG-HA spongy-like hydrogels. Moreover, a higher CD163+/CD86+ macrophages ratio was obtained for all the conditions, significantly higher for GG-HA materials with hASCs pre-cultured in neurogenic medium, revealing a switch from the inflammatory to the proliferative phase of wound healing. Neoinnervation was achieved with unconditioned stem cell-laden GG-HA spongy-like hydrogels but not with conditioned stem cells. Altogether the results obtained under the scope of this thesis gave interesting insights on the effects of the new GG-based matrices over mechanisms that are pivotal for wound healing. Furthermore, GG-based matrices, especially if used as part of an integrated approach that targets the different pathophysiologies of diabetic wounds, represent a promising approach to positively impact reepithelialization and modulate the inflammatory response towards its resolution, which seems to be determinant for a successful neoinnervation.
A elevada prevalência da Diabetes no Mundo está a fazer aumentar a incidência de úlceras do pé diabético. As várias fisiopatologias das feridas diabéticas, nomeadamente a re-epitelização atrasada e imatura, a inflamação persistente, a isquemia e a neuropatia, atrasam a cicatrização das feridas que facilmente evoluem para úlceras. A problemática deste tipo de feridas reside no tempo de cicatrização que em muitos casos pode demorar meses ou mesmo não cicatrizar, resultando na amputação do membro afetado. Deste modo, existe uma necessidade premente de um tratamento eficaz para as úlceras diabéticas. A capacidade de os hidrogéis absorverem e reterem água tem sido reconhecida como extremamente valiosa na manutenção da humidade em feridas. Para além disso, os hidrogéis per se, ou incorporando diferentes células e/ou agentes bioativos, têm originado respostas biológicas benéficas. Nesta tese, postulou-se uma estratégia que envolvesse o uso de hidrogéis com polímeros bioativos ou células estaminais direccionanda para o tratamento das patologias das feridas diabéticas. Os hidrogéis de goma gelana (GG) têm sido vastamente estudados para diferentes aplicações biomédicas. No entanto, a baixa resistência a deformações, a falta de capacidade para promover a adesão e aquisição da morfologia nativa das células no seu interior, e a falta de biodegradação, não são ideais para tratar feridas de pele. O primeiro objetivo desta tese foi superar estas limitações. A ausência de características que permitam a adesão e aquisição de morfologia nativa das células foi superada recorrendo a duas estratégias: o rearranjo microestrutural dos hidrogéis de GG, originando hidrogéis esponjosos de GG, e a adição química de sequências peptídicas, promotoras de adesão celular, ao polímero de GG previamente modificado com divinil sulfona (GGDVS). A biodegradação foi alcançada intercalando um polímero degradável, o ácido hialurónico (HA), na estrutura reticulada de GG, assim como utilizando um agente de reticulação sensível à ação de metaloproteases. Estas duas matrizes à base de GG adquiriram uma estabilidade mecânica superior aos hidrogéis de GG que também pôde ser modulada variando o agente reticulante e a quantidade do polímero. As propriedades individuais de cada uma das matrizes à base de GG são igualmente relevantes para o tratamento de feridas da pele. Os hidrogéis esponjosos de goma gelana podem ser mantidos e armazenados no estado seco que, após serem hidratados, re-adquirem a forma de um hidrogel esponjoso. Para além disso, o custo de produção destes materiais é relativamente baixo e podem ser produzidos sob qualquer forma e tamanho, de acordo com as especificidades da ferida. Estes materiais prontos a usar podem ser utilizados na sua forma desidratada para absorver o exsudado da ferida, ou hidratados com uma solução aquosa com/sem células ou agentes bioactivos, imediatamente antes da aplicação na ferida. Para além de poderem transportar células aderentes ou agentes bioactivos, os hidrogeis modificados GGDVS têm a vantagem de serem injetáveis, um fator de extrema importância para o tratamento de feridas interdigitais. Num contexto de cicatrização de feridas da pele, abordamos o efeito da libertação sustentada de eumelanina (extraída de Sepia officinalis) a partir de hidrogéis esponjosos de GG sobre queratinócitos. Foi demonstrada a capacidade dos queratinócitos humanos primários fagocitarem a eumelanina, que se acumulou tanto em vesículas do citoplasma como também à volta do núcleo. A viabilidade e maturação dos queratinócitos não foi afectada pelo contacto com a eumelanina, no entanto, a morfologia e a proliferação das células foram alteradas de um modo dependente da concentração. Por fim, a eumelanina, previamente internalizada por queratinócitos, reduziu as espécies reactivas de oxigénio libertadas após a exposição dos queratinócitos a radiação UVA/UVB, de um modo dependente da concentração e do tempo de exposição. Deste modo, os efeitos da eumelanina sobre os queratinócitos podem ser vantajosos para beneficiar a cicatrização das feridas da pele. A possibilidade de conferir um carácter angiogénico à matriz esponjosa de GG foi investigada através de duas estratégias: a intercalação de HA nas redes poliméricas de GG (GG-HA) e a modificação do GG com o grupo divinil sulfona e os péptidos de adesão T1 e C16, das proteínas CCN1 e laminina, que podem ser reconhecidos por células endoteliais. Os hidrogéis esponjosos contendo HA tornaram-se susceptíveis à biodegradação pela hialuronidase com consequente formação de fragmentos de HA de baixo peso molecular, que demonstraram ser capazes de melhorar a perfusão sanguínea e aumentar a neovascularização num modelo animal de isquemia. Apesar do potencial angiogénico dos péptidos T1 e C16 não ter sido estudado, as células endoteliais aderiram no interior dos hidrogeis modificados com os péptidos, de um modo dependente do tipo e quantidade de péptido. Enquanto que a morfologia das células endoteliais no interior dos hidrogéis com C16 aparentou ser mais arredondada, nos hidrogéis com T1 foi mais polarizada. Tendo em conta os resultados anteriores, uma estratégia que concomitantemente modulasse a inflamação e angiogénese, e que promovesse a neoinervação, foi considerada. De modo a aumentar o secretoma que potencia a regeneração nervosa, células estaminais do tecido adiposo foram precultivadas em meio neurogénico ou normal no interior de materiais esponjosos de GG-HA e transplantadas para as feridas diabéticas de um modelo animal. Relativamente ao controlo (sem material), houve um aceleramento do fecho da ferida e re-epitelização, assim como uma maior proporção de macrófagos com expressão CD163+/CD86+, de um modo significativo nas feridas tratadas com hidrogéis esponjosos contento células estaminais pré-condicionadas em meio neurogénico, indicativo de uma progressão da fase inflamatória para a fase proliferativa da cicatrização da ferida. Uma epiderme mais espessa e completamente maturada foi observada nas feridas tratadas com hidrogéis esponjosos contendo células estaminais. Por fim, uma maior neoinervação foi observada para a condição contendo células estaminais, contrariamente à condição contendo células estaminais condicionadas. Em suma, os resultados obtidos no âmbito desta tese forneceram indicações interessantes sobre os efeitos das novas matrizes de GG sobre mecanismos essenciais para a cicatrização de feridas. Do mesmo modo, as matrizes de GG, especialmente se usadas como parte de uma abordagem integrada direccionada para o tratamento das diferentes patofisiologias de feridas diabéticas, representam uma abordagem promissora capaz de promover positivamente a cicatrização de feridas, não só ao nível da re-epitelização, mas também modulando a resposta inflamatória promovendo a sua resolução, um efeito que aparenta ser determinante para uma neoinervação bem sucedida.

碩士
靜宜大學
食品營養學系
89
ABSTRACT The purpose of this study was to investigate the influences of starch granule, concentration and variety on the rheological properties of polysaccharide solutions and gels. For the mixtures of red algal polysaccharides with starches, the apparent viscosities increased linearly with increasing starch concentrations. The increasing effects of added starch components were greater for gelatinized starches than for native starches. The storage (G¢) and loss (G²) moduli as well as shear thinning properties of the mixtures except l-carrageenan-containing systems rose in the presence of starches (e.g. corn, potato and cassava starches) in either native or gelatinized state. When mixed with konjac flour, the systems of red algal polysaccharides (except for κ-carrageenan systems) with a lower molecular weight showed a greater Tgel and lower moduli on gelation by cooling. After aging at room temperature for one day, the dynamic rheological properties of the mixed polysaccharide-starch systems were examined. Pure gelatinized starch and l-carrageenan systems showed G¢ and G² maxima at ~50oC. However, the moduli of their mixtures with gelatinized corn starch decreased gradually at > 30oC. Rheological properties of weak gels were observed in the l-carrageenan-gelatinized starch mixtures. In the case of konjac flour-starch mixtures, the increasing effects of added native (20%) or gelatinized (3%) starch components on the apparent viscosities of the mixed polysaccharide solutions appeared to be greater for the starches with high amylose contents. All mixtures containing konjac flour behaved as concentrated solutions with shear thinning properties. On cooling of 1% konjac flour and gelatinized corn starch systems, the observed moduli increased linearly without notable phase transitions on gelation. After aging for 24 h and subsequent reheating on dynamic rheological measurements, the gel characteristics of these systems remained at up to 50oC, different from the results of pure konjac flour systems. As for the mixtures of other polysaccharide gums with starches, the increasing effects of gelatinized starches on the apparent viscosities of the mixed solutions were 12-75 times those of native starches, depending on the variety and concentration of gums and starches concerned. The addition of native starches did not significantly change the dilute-solution properties of guar gum (GG), locust bean gum (LBG), carboxymethyl cellulose (CMC) and gum arabic systems. This also held for the gum arabic or CMC mixtures with gelatinized starches. The appearance of gelatinized starches resulted in the weak-gel properties of GG and gellan gum. Nonetheless, the elastic-gel properties of gellan or xanthan gums were hardly influenced by the added starches. On reheating the above mixtures after aging, pure GG showed G¢ and G² maxima at ~52oC, accompanying with a minimum in tan d. The critical temperature was greater than those for the GG mixtures with starches (~35oC). Similar phenonema to the pure GG systems were also observed in pure CMC and xanthan gum systems. However, the G¢ and G² of CMC or xanthan gum with starches decreased successively with increasing reheating temperature. The moduli of pure gum arabic or its mixtures with starches increased significantly at > 35oC and maximized at ~45oC.

碩士
靜宜大學
食品營養研究所
86
Effects of gum concentration (0.3 - 1.8%), starch types (wheat, corn and cassava) and concentration (2-4%) on the gelling behavior of Hsian-tsao (Mesona procumbens Hemsl) leaf gum extracted by sodium carbonate solutions were studied. Steady and dynamic shear rheological measurements, rapid visco analysis, texture profile analysis, color as well as microstucture of the starch/gum mixtures were performed during or after a controlled heating and cooling process. It is found that the starch/gum mixture showed higher rheological/textural properties than starch or gum alone at comparable concentration, indicating significant interactions between starch and Hsian-tsao leaf gum. The gel viscosity as determined by rapid visco analysis, storage modulus as determined by dynamic shear measurement, apparent viscosity as determined by steady shear measurement, as well as firmness as determined by texture profile analysis, of the mixed system increased with increasing gum concentration to a certain extent, then leveled off or decreased with higher gum concentration. The critical gum concentration, which provide an optimum interaction with starch, depended on starch types and increased with increasing starch concentration. The mixed systems showed typical biopolymer gel behavior since their tan delta values were found to be less than one or close to one. In addition, the mixed systems exhibited shear thinning behavior since their apparent viscosity decreased with increasing shear rate. A power law model was used to simulate the flow behavior of the mixed systems. The flow behavior index in the power law model was found to decrease, whereas, the consistency index increase with increasing gum concentration in the mixed systems. A comparison between the steady shear and dynamic shear rheological properties of the mixed system showed that Cox-Merz superposition principle was not suitable for the Hsian-tsao gel system. In general, mixed systems containing wheat or corn starch were brighter than the one containing cassava starch, as indicated by the Hunter''s L values. Scanning electron microscopy observation revealed that a denser and ordered network was formed as the amount of starch and gum increased to a certain extent.