Orientador: Vera Aparecida de Oliveira Tiera / Banca: Éder Tadeu Gomes Cavalheiro / Banca: Marcelo de Freitas Lima / Resumo: Os fungos do gênero Aspergillus, presentes em amendoins, milho, arroz, nozes e outros grãos, são uma constante preocupação nos dias atuais devido a sua capacidade de produzir toxinas cancerígenas. Dentre as várias técnicas de controle desse microrganismo, tem se destacado o uso de produtos naturais, não agressivos ao meio ambiente e de baixo custo, como por exemplo, a quitosana. O presente trabalho apresenta a síntese e caracterização de duas séries de novos derivados de quitosana para utilização como biofungicida contra os fungos Aspergillus flavus e Aspergillus parasiticus. A primeira série descreve a síntese de derivados de quitosana pela reação de quitosana desacetilada (QD) com brometo de propil (Q-Propil) pentil (Q-Pentil) trimetilamônio para obter derivados com proporções crescentes do grau de substituição (GS). A segunda série foi obtida a partir da síntese de sais bi-quaternários de amônio, com a posterior reação com quitosana desacetilada obtendo-se derivados bi-quaternários de quitosana. Todos os derivados foram caracterizados por técnicas de RMN 1 H, infra-vermelho (IR) e titulação potenciométrica e as suas atividades fungicidas testadas contra o crescimento micelial dos fungos A. flavus e A. parasiticus in vitro. Os estudos foram conduzidos variando-se a acidez e a força iônica do meio. Os resultados dos ensaios microbiológicos mostram que as atividades antifúngicas aumentam com o grau de substituição e os derivados mais substituídos de ambas as series, propil e pentil, exibiram respectivamente atividades três e seis vezes maiores que as quitosanas comercial e desacetilada. As concentrações inibitórias mínimas foram avaliadas após 24, 48 e 72 horas, variando-se a concentração dos polímeros de 0,5 a 16 g L -1 e os resultados mostraram que os derivados quaternários inibem o crescimento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The fungi of the genus Aspergillus, present in peanuts, corn, rice, nuts and other grains, are a constant concern nowadays due to its ability to produce carcinogenic toxins. Among the various techniques for controlling this microorganism it has been emphasized the use of natural products not harmful to the environment and at a low cost, for example, chitosan. This paper presents the synthesis and the characterization of two series of novel derivatives of chitosan for use as biofungicide against the Aspergillus flavus and Aspergillus parasiticus fungi. The first series of derivatives was synthesized by the reaction of deacetylated chitosan (QD) propyl bromide (Q-Propil) and pentyl trimethylammonium (Q-Pentil) for obtaining derivatives with increasing degrees of substitution (GS). The second series was obtained from the synthesis of bi-quaternary salts, with subsequent reaction with deacetylated chitosan to yield bi-quaternary derivatives of chitosan (Q-BPHD). All derivatives were characterized using NMR techniques, infra-red (IR) and potentiometric titration and fungicides activities were tested against A. flavus and A. parasiticus by evaluating the mycelial growth of fungi in vitro. The studies were conducted by varying the acidity and ionic strength of the medium. The antifungal activities increase with the degree of substitution and the most substituted derivatives from both series, propyl and pentyl, respectively exhibited activities three to six times lighter than the commercial and deacetylated chitosans. The minimum inhibitory concentrations were recorded after 24, 48 and 72 hours varying the concentration of polymer from 0.5 to 16 g L -1 and the results showed that the quaternary derivatives inhibit the growth of fungi in concentrations four times lower than deacetylated chitosan . The bi-quaternary derivative (Q-BPHD) showed the... (Complete abstract click electronic access below) / Mestre

Os filmes de quitosana/amido são utilizados para preparação de sistemas biocompatíveis de liberação controlada, materiais de revestimento e filmes comestíveis. As propriedades reológicas, térmicas, mecânicas, de taxa de transmissão de vapor de água (WVP) e solubilidade foram avaliadas em função das mudanças estruturais do amido (gelatinizado ou oxidado), da adição de polióis (etilenoglicol, glicerol e sorbitol) e da razão amilose/amilopectina (73% e 100% amilopectina). Os ensaios de reologia mostraram que as blendas formadas por quitosana e amido (100% amilopectina) tiveram interações mais fortes quando comparadas com as blendas de quitosana/amido (73% amilopectina), tanto na forma oxidada quanto na forma gelatinizada. Os polióis causam mudanças nas redes tridimensionais formadas entre a quitosana e o amido, tanto quando se usa amido gelatinizado como quando se usa o amido oxidado. Os valores de G´ encontrados para as blendas preparadas com amido oxidado foram em todos os casos menores em relação às blendas preparadas com amido gelatinizado, indicando diferenças na estrutura do gel formado e também na organização dos seus componentes. Essas mudanças foram resultantes da associação que ocorre entre a quitosana e o amido oxidado que é diferente daquela que ocorre entre quitosana e amido gelatinizado. A oxidação do amido A termogravimetria mostrou que a razão amilose/amilopectina não influencia o perfil de perda de massa e a oxidação amido aumenta a quantidade de água nos filmes e também diminui a estabilidade térmica observada por Tonset. O glicerol é o poliol que provoca maiores mudanças no comportamento de perda de massa dos filmes, pois é observada a decomposição de parte do glicerol que não está interagindo com a quitosana e o amido. O DSC mostrou que a presença de amilose nas blendas provoca uma interação mais forte da água com os polissacarídeos. Para todos os filmes com amido gelatinizado, a adição de polióis aumenta a temperatura de pico endotérmico e esta é quase sempre maior quando o glicerol é adicionado e que a oxidação do amido provoca um aumento nos valores de pico endotérmico, sugerindo que mais água está interagindo com os polímeros. A adição dos polióis aumenta os valores de espessura dos filmes, pelo aumento do volume livre do sistema, tanto nos filmes preparados com amido gelatinizado, quanto no oxidado. A presença de amilose nos filmes aumenta os valores de WVP e de solubilidade, provavelmente porque a presença de amilose auxilia no aumento da difusão de moléculas de água pelo e facilita a entrada de água e sua posterior solubilização. A adição dos polióis diminui os valores de WVP nos filmes de quitosana e amido gelatinizado. A oxidação do amido interfere mais nos valores de WVP para os filmes de quitosana/amido (100% amilopectina), sugerindo maior difusão das moléculas de água pelo filme. A oxidação aumenta os valores de solubilidade, pois as interações ocorrem em menor quantidade entre a quitosana e o amido, o que acaba facilitando o processo de solubilização. A adição dos polióis provoca um aumento na solubilidade de todos os filmes. Os ensaios preliminares de tração por DMA indicaram que os filmes de quitosana/amido gelatinizado e os filmes de quitosana/amido oxidado são rígidos e quebradiços e com a adição dos polióis ocorre um aumento na flexibilidade. Estudos preliminares destes filmes como suportes para liberação controlada de ciprofloxacina mostraram que estes são promissores para esta aplicação. / Chitosan/starch association represents an interesting alternative to the preparation of biocompatible drug delivery systems, packing materials and edible films. Changes in rheological, thermal and mechanical properties, water vapor permeability and solubility of chitosan/starch films were evaluated varying the starch structure form (gelatinized or oxidized), poliol addition (ethyleneglycol, glycerol and sorbitol) and amylose/amylopectin ratio (73% and 100% amylopectin). Rheological measurements showed that chitosan/starch (100% amylopectin) blends had stronger interaction compared to chitosan/starch (73% amylopectin) blends, both in gelatinized and oxidized starch form. Polyols cause changes in chitosan/starch three-dimensional networks due to reduction in polymer-polymer chain bonding that reflect in rheological properties. G´ modulus value decreases with starch oxidation, indicating variation in gel structure and also in organization of polymeric components, due to differences in association between chitosan and gelatinized or oxidized starch. Thermogravimetric curves showed that amylose/amylopectin ratio does not influence the weight loss behavior and starch oxidation increases the amount of water and decrease thermal stability of films, as observed by Tonset. Glycerol causes changes in weight loss behavior due to the presence of glycerol that not interact with chitosan and starch. DSC results suggest that amylose presence promotes a higher water interaction with polysaccharides. Polyol addition shift the endothermic peak associated to free water for higher temperatures, suggesting interaction between biopolymers and polyols. Also, polyol increases thickness values in all films as increase the free volume of the system. Amylose presence increases WVP and solubility values, suggesting the formation of low dense structure, which increases the water molecules diffusion and facilitates the dissolution of films in water. Polyols presence decrease WVP values in chitosan/gelatinized starch films. Starch oxidation affected the WVP values in chitosan/starch (100% amylopectin) films, suggesting low compact structure that allows the water molecule diffusion in the films. Starch oxidation increases solubility values as lower interaction between chitosan and oxidized starch, which facilitates the solubility process. Polyols addition causes an increase in solubility in all films. Preliminary mechanical tests performed by DMA indicated that chitosan/starch (gelatinized or oxidized) films are rigid and brittle and polyol addition increases the flexibility. In vitro ciprofloxacin drug delivery behavior showed that films are promising for this application.

A quitosana é um biopolímero estudado em diversas áreas, tais como, ambiental, alimentícia, farmacêutica, biomédica e biotecnológica. Ela pode ser obtida de diferentes formas polimórficas de quitina, dentre as quais a forma β tem se mostrado vantajosa, pois favorece modificações químicas mais homogêneas e leva a um produto final menos alergênico. A quitosana pode ser combinada com outros compostos a fim de interagir e/ou reagir com eles e modificar suas propriedades. O objetivo deste trabalho foi estudar como uso de ácido adípico, em substituição ao acético, afeta as propriedades de géis e esponjas de quitosana e de quitosana/gelatina, que foram posteriormente reticuladas com os agentes reticulantes EDC/NHS. As técnicas utilizadas para os estudos foram: reologia, FTIR, MEV, absorção em PBS e ensaios de citotoxicidade. Por reologia, observou-se que o aumento na concentração dos géis de quitosana tornou os géis mais elásticos e viscosos. O mesmo ocorreu na presença de gelatina ou EDC/NHS. O efeito do uso de ácido adípico em substituição ao acético também foi mostrado nos ensaios reológicos, pois os géis com 2% de quitosana e com quitosana/gelatina sem EDC/NHS se mostraram mais elásticos e mais viscosos quando o ácido adípico foi usado. Os espectros FTIR mostraram a presença de interações entre a quitosana e a gelatina e a formação de ligações amidas II após reticulação com EDC/NHS. Na preparação das esponjas observou-se que os géis de quitosana em ácido adípico geravam esponjas instáveis que se desfizeram durante a neutralização, mas essa instabilidade não ocorreu com a blenda. As esponjas preparadas com a blenda foram estudadas após neutralização e o MEV mostrou que o uso de EDC/NHS alterou a morfologia levando a formação de poros interconectados. Nos ensaios de absorção em tampão de PBS foi observado que o uso de ácido acético aumenta a absorção para as esponjas sem EDC/NHS, enquanto para as esponjas com EDC/NHS a absorção é maior quando se usa ácido adípico. Todas as esponjas foram não citotóxicas o que torna esses materiais promissores para serem estudados em aplicações na área médica, tais como material de curativo, implantes, liberação controlada de fármacos. / Chitosan is a natural polymer studied in various fields such as environmental, food, pharmaceutical, biomedical and biotechnology. It can be obtained from different polymorphic forms of chitin, of which the form β has proven advantageous because it promotes more homogeneous and chemical modifications leads to a final product less allergenic. Chitosan can be combined with other compounds and thus further improve its properties. The aim of this study was to analyze how the use of adipic acid, replacing acetic acid affects the properties of gels and sponges of chitosan and chitosan/gelatin, which were subsequently crosslinked with EDC/NHS. The techniques used for these studies were: rheology, FTIR, SEM, absorption in PBS and cytotoxicity assays. In rheology, it was observed that increasing the concentration of chitosan was possible to prepare more elastic and viscous gels. The same occurs in the presence of gelatin or EDC/NHSO. The effect of the use of adipic acid to replace the acetic acid was also shown on rheological measurements, because the gels with 2% chitosan or chitosan/gelatin without EDC/NHS were more elastic and more viscous when the adipic acid has been used. The FTIR spectra showed the presence of interactions between chitosan and gelatin and the formation of amide II Bonds after crosslinking with EDC/NHS. In the preparation of the sponges it was observed that the gels of chitosan with adipic acid generated unstable sponges crumbled during neutralization, but this instability does not occur with the blend. Sponges prepared with the blend were studied after neutralization and SEM showed that the use of EDC/NHS altered the morphology leading to the formation of interconnected pores. The use of acetic acid increases the absorption in PBS for sponges without EDC/NHS, while for sponges with EDC/NHS the absorption is greater when adipic acid was used. All sponges were non-cytotoxic making them promising materials to be studied for applications in the medical field, such as dressing materials, implants, controlled drug release.

Uma variedade de hidrogéis, que correspondem a uma classe de materiais poliméricos altamente hidratados está sendo empregado como biomateriais. Biopolímeros como quitosana, xantana e colágeno apresentam excelente biocompatibilidade e podem ser utilizados na área de queimados e liberação controlada de fármacos. Neste trabalho foram preparados hidrogéis provenientes da mistura entre quitosana, derivada de β-quitina, xantana comercial e colágeno aniônico (derivado de tendão bovino) em proporções variadas. A caracterização das amostras foi realizada por análise térmica (termogravimetria e calorimetria exploratória diferencial), espectroscopia no infravermelho, microscopia eletrônica de varredura e absorção de água. Pelas curvas termogravimétricas os resultados mostraram que todos os hidrogéis apresentam uma grande quantidade de água na sua estrutura e que pela reidratação são capazes de reter novamente essa quantidade de água. Por DSC observou-se que todos apresentaram o pico referente à fusão de água livre em temperaturas deslocadas acima de 0oC pelo aumento da interação água/polímero. Por FT-IR constatou-se a presença de bandas características dos biopolímeros com a ausência de novas bandas e pelo estudo por MEV os hidrogéis apresentaram uma estrutura na forma de folhas e a presença de colágeno é bastante visível participando desta estrutura. / Hydrogel are polymeric materials, which have the capacity to retain a great amount of water, and are often used as biomaterials. Chitosan, xanthan and collagen present biocompatibility and they can be used in tissue regeneration and drug delivery. In this work hydrogels were prepared by the mixture with chitosan (from β-quitina), commercial xanthan and anionic collagen (from bovine tendon) in different proportions. They were characterized by thermal analysis (thermogravimetry and differential scanning calorimetry), infrared spectroscopy (FTIR), scanning electronic microscopy and water absorption. Thermogravimetric curves showed that all hydrogels present a great amount of water and after the rehydratation were capable to keep the same amount of water. Melting point of water in DSC curves were shifted to higher values due the increase of water/polymer interaction. FT-IR showed characteristic bands of biopolymers with the absence of new bands and the morphology (MEV) showed that the hydrogels structures were in sheet form and the collagen fibers are visible in the hole structure.

Philosophiae Doctor - PhD / The fluoride release and chemical adherence to tooth structure remain the most desirable features of glass ionomer restorative cements (GICs). Although the physical properties for multi-surface restorations are well-defined, even with the introduction of newer GICs not all demands have been met. Yet, increased use of GICs will only be possible if clinicians change their perceptions of the low survival rate of GICs. The lower clinical success rate of GICs is partly due to the marginal integrity and wear over time, which has often been recorded in the literature as restoration failure. The current, well-established restorative options for the primary dentition are Resin Modified Glass Ionomers (RMGICs) and Compomer resins. There is a paradigm shift towards materials that are more biologically favourable. Areas of research for dental materials include antibacterial properties in conjunction with ion release to maintain healthy restored teeth. If a GIC can provide adequate physical properties with the inclusion of the aforementioned features, GICs might become a more viable permanent restorative solution.

Soluble surimi wash water (SWW) proteins could be recovered using chitosan (Chi) complexed with alginate (Chi-Alg) generating co-products for feed formulations. Chi with a degree of deacetylation (DD) of 84% complexed with Alg at a mixing ratio (MR) of 0.2 was used to study Chi-Alg concentration and treatment time protein recovery effects. Insoluble SWW solids were removed by centrifugation and the supernatant was then adjusted to pH 6. Flocculation at 20��C using Chi-Alg at 20, 40, 100 and 150 mg/L SWW was aided by 5 mm agitation and holding for 30 mm, 1h and 24h. Concentration had an effect between low (20 and 40 mg/L) and high (100 and 150 mg/L) levels. Time had an effect between 30 min and 1h but not between 1 and 24 h. Turbidity reduction was affected only by concentration. 100 mg Chi-Alg/L SWW for 1 h achieved 83% protein adsorption and 97% turbidity reduction while lower concentrations yielding higher adsorption required longer times. Fourier Transform Infrared (FTIR) analysis of untreated and Chi-Alg treated SWW solids confirmed protein adsorption. Amide band areas normalized against a common 3005-2880 cm����� region confirmed the high protein recovery by 100 mg Chi-Alg/L SWW. Six Chi samples differing in molecular weight (MW) and degree of deacetylation (DD) were tested to recover soluble SWW solids using 20, 40, and 100 mg Chi-Alg/L SWW (0.2 MR, 1h). High (94%, 93%) and low (75%) DD chitosan had lower protein adsorption (73-75%) when compared to the intermediate (84%) DD chitosan (74-83%). Intermediate DD and high MW Chi seemed to perform better; however, SY-1000 with 94% DD did not follow this trend (79-86% protein adsorption, 85-92% turbidity reduction). Insoluble SWW (P1) and soluble solids (P2) recovered using 150 mg Chi- Alg/L SWW contained 61.4 and 73.1% protein, respectively. Rat diets formulated with 10% protein substitution by P1 and 10% and 15% by P2 had acceptability and protein efficiency ratios (PER) as high as the casein control with no deleterious effects. Rat diets with 100% P2 protein substitution showed higher PER and net protein ratio than the casein control with no deleterious effects. Protein recovered from SWW using Chi-Alg has the potential to be used in commercial feed formulations. / Graduation date: 2004

Graduation date: 1996

Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007

Chitosan is known to be antibacterial and antifungal, but information on its effectiveness against foodborne viruses is limited. Enteric viruses are a major concern in food safety, especially human noroviruses which are the leading cause of nonbacterial gastroenteritis. The overall goal of this research was to determine the antiviral effectiveness of chitosan. The specific objectives were to determine the effects of molecular weight (MW) and concentration of chitosan against the cultivable enteric viral surrogates, feline calicivirus (FCV-F9), murine norovirus (MNV-1), and bacteriophages (MS2 and phiX174). Purified chitosans (53, 222, 307, 421, ~1,150kDa) were dissolved in water, 1% acetic acid, or aqueous HCl (pH= 4.3), and sterilized by membrane filtration. The solutions were mixed with equal volume of virus suspension to obtain a virus titer of 5 log PFU/ml and chitosan concentration of 0.7% for all five MW and 0.7, 1.0, 1.25, and 1.5% for 53 and 222kDa. The samples were incubated for 3 hr at 37°C before viral enumeration. Controls included untreated viruses in PBS, in PBS with acetic acid, and in PBS with HCl. Chitosan showed the greatest reduction of MS2, followed by FCV-F9, phi X174, and MNV-1. A MW effect was seen with MS2, with higher MW being more efficient, and 0.7% of ~1,150kDa causing complete inactivation. Increasing the concentration of chitosan from 0.7 to 1.5% reduced the titer of MS2 and FCV-F9 by 5.16 and 2.91 logs, respectively. Although chitosan was ineffective against MNV-1, its ability to significantly reduce MS2 and FCV-F9, suggest its use for future foodborne viral control.

In scaffold-based tissue engineering, control of scaffold degradation turns out to be a critical issue for reliable clinical applications. Degradation in this thesis refers to mass loss. Most of the present control methods take the approach of scaffold material modification and/or scaffold work environment adjustment to address this issue. The latter can easily get to its limit, and the former is not promising in the in-vivo implementation. This thesis proposed a new approach to control of scaffold degradation, that is, closed-loop and real-time control. To realize this approach, this thesis has tackled three important problems, namely (1) effects on degradation, (2) modeling of degradation, and (3) real-time measurement of degradation. This thesis is grounded to a biomaterial called chitosan, as it is widely used for building scaffolds. For the first problem, a statistical experiment was designed and a factorial analysis was conducted. For the second problem, a combined empirical-based and probabilistic-based approach was taken. For the third problem, a prototype of a sensor, which is based on the concept of carbon nanotube (CNT) conductive polymer, was built and tested. This thesis concludes (1) a joint thermal and enzymatic effect is significant on chitosan degradation, (2) the model for chitosan degradation is accurate, and (3) real-time measurement of mass loss of scaffold by means of carbon nanotube film is feasible. The major contributions of this thesis are (i) the proposal of the concept of the closed-loop control of degradation, (ii) a finding that there is a significant conjoint thermal and enzymatic effect on chitosan degradation in terms of mass loss, and (iii) a prototype of the novel CNT (carbon nanotube) chitosan film sensor for real-time measurement of mass loss of the scaffold. The significance of these contributions is that they give us confidence to a full development of the closed-loop and real-time degradation control approach. This approach appears promising to bring forth a transformative impact to clinic applications of scaffold-based tissue regeneration.