The field of tissue engineering aims to develop viable substitutes with the ability to repair and regenerate the functions of damaged tissue. Common practices to supplement bone regeneration in larger defects include bone graft biomaterials such as autografts, allografts, xenografts, and synthetic biomaterials. Autologous bone grafting is the current gold-standard procedure used to replace missing or damaged bone. However, these grafts have disadvantages such as donor site morbidity, limited availability, and the need for a secondary surgery. The focus of this study is to tissue engineer a lyophilized gelatin composite sponge composed of hydroxyapatite (HA), chitin whiskers (CW), and preparations rich in growth factors (PRGF) to provide sufficient structural support to the defect site while enhancing the body’s own reparative capacity, ultimately eliminating the need for autologous tissue harvesting or repeat operations. The present study investigates several in vitro evaluations on multiple compositions of modified gelatin sponge scaffolds for use in bone graft applications. Gelatin sponges were fabricated via freeze-drying, enhanced with PRGF, HA, and/or CW, and cross-linked with 50 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) either during or post gelation. Initial evaluation of all scaffold combinations indicated that scaffolds released contents up to 90 days, EDC cross-linking during gelation allowed for more protein release, and had the ability to swell. Since the incorporation of PRGF, HA, and CW increased cell infiltration, and production of cell-created mineral matrix over 90 days in culture, these scaffolds were further characterized. Freeze-dried gelatin sponges enhanced with PRGF, HA, and CW and cross-linked during gelation with EDC (PHCE) were mineralized (M) in 5x revised simulated body fluid (r-SBF) for 1 hour to create a bone-like mineral surface. Gelatin EDC scaffold controls (GE), GE-M, PHCE, and PHCE-M scaffolds were characterized for their ability to swell, mineralizing potential, surface morphology, growth factor incorporation and release, uniaxial compression properties, and cell attachment, proliferation, infiltration, and protein/cytokine secretion.. After mineralization, scanning electron microscopy showed sparse clusters of mineral deposition for GE-M scaffolds while PHCE-M scaffolds exhibited a more uniform mineral deposition. Both GE and PHCE scaffolds were porous structures that swelled up to 50% of their original volume upon hydration. Over 21 days incubation, PHCE-M scaffolds cumulatively released about 30% of their original protein content, significantly more than all other scaffolds. Multiplex Luminex assays confirmed the successful incorporation of PRGF growth factors within PRGF sponges. For acellular uniaxial compression testing, PHCE-M scaffolds reported lower Young’s modulus values (1.3 - 1.6 MPa) when compared to GE and GE-M scaffolds (1.6 – 3.2 MPa). These low modulus values were comparable to values of tissue found in early stages of bone healing. DAPI (4',6-diamidino-2-phenylindole) staining and imaging showed an increase in initial cell attachment and infiltration of PHCE and PHCE-M scaffolds on day 1. GE-M scaffolds also appeared to attach more cells than the GE control. MTS cell proliferation assay results indicated that on days 4 and 7, PHCE scaffolds increased cell proliferation (compared to GE controls). MTS also illustrated that the addition of a mineral coating increases and decreases cell proliferation on GE-M and PHCE-M scaffolds, respectively. Multiplexer analysis of MG-63 protein/cytokine secretion suggests that cells are responding in a bone regenerative fashion on all scaffolds, as evidence of osteocalcin secretion. Little to no secretion of osteopontin, IL-1β, and TNF-α demonstrates that scaffolds are not influencing cells to secrete factors associated with bone resorption. The compressive mechanical properties of cellularized scaffolds did not differ much from acellular scaffolds. The collective results indicated increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion by cells on GE-M scaffolds, which support the addition of a bone-like mineral surface on GE scaffolds. Cellularized PHCE and PHCE-M scaffolds report similar advantages as well as Young’s modulus values in the range of native tissues present in the early stages of bone healing. The results of this study propose that the developed PHCE and PHCE-M scaffolds exhibit good cellular responses and mechanical properties for use in early bone healing applications.

La chitine est l’un des biopolymères les plus abondants dans la biomasse. Son principal dérivé industriel est le chitosane. Ces deux polysaccharides présentent un intérêt croissant en raison de leurs nombreuses propriétés physico-chimiques et biologiques remarquables. Leur utilisation en tant que matériaux est potentiellement intéressante dans de nombreux domaines tels que la pharmacie, la médecine, l’industrie alimentaire et l'agriculture. Malgré de nombreuses avancées dans les méthodes chimiques de production de la chitine et du chitosane, l'utilisation de solutions concentrées d'acides et de bases alcalines présente le désavantage de générer de grandes quantités d’effluents toxiques. Récemment, des méthodes d'extraction de la chitine par biotechnologie ont été proposées comme une alternative aux méthodes chimiques. Dans ce contexte, l'objectif principal de ce travail a été de développer un procédé biotechnologique d’extraction de la chitine à partir de carapaces de crustacés à l’aide de bactéries lactiques et d’enzymes. A cette fin, les facteurs influençant les réactions mises en jeu au cours de l’extraction, telles que la déminéralisation, la déprotéinisation et la N-désacétylation ont été étudiés en considérant les caractéristiques physico-chimiques des chitines et chitosanes ainsi obtenus. Ces caractéristiques sont principalement les masses molaires moyennes et le degré de N-acétylation. Ce travail s’est également intéressé à la valorisation des sous-produits d’extraction (protéines, pigments…) et aux applications potentielles des différents chitosanes préparés, notamment comme agents fongistatiques. / The chitin is one of the most abundant biopolymers in biomass. Its main industrial derivative is the chitosan. These two polysaccharides present an increasing interest thanks to their various interesting physicochemical and biological properties. Their potential applications concern diverse fields as the pharmacy, medicine, food industry and agriculture. Despite numerous advances in methods for the chemical production of chitin and chitosan, the use of concentrated solutions of acids and alkaline bases presents the disadvantage to generate large amounts of toxic wastes. Recently, biotechnological methods of chitin extractions have been proposed as an alternative to chemical methods. In this context, the main objective of this work was to develop a biotechnological process for the extraction of chitin from crustacean shells using lactic acid bacteria and enzymes. For this purpose, factors influencing reactions involved in the extraction, i.e. the demineralization, deproteinization and N-deacetylation, were studied considering the physicochemical characteristics (molecular weight and degree of N-acetylation) of chitin and chitosan produced. The recovery of extraction side products such as proteins and pigments has also been considered in this project, as well as the evaluation of various chitosans prepared by this process as fungistatic agents.

Shellfish waste obtained from seafood processing plants contains chitin, protein and calcium carbonate. Chitin is a versatile biopolymer with many applications. Conventionally, chitin is separated from calcium carbonate and protein by acid and alkali respectively. In this project, a biotechnological approach was applied to recover chitin from scampi (Nephrops norvegicus) waste using lactic acid bacteria (LAB) to produce lactic acid from glucose which lowers the pH of the mixture, thus preserving the waste from spoilage. The acid also dissolves the calcium carbonate and under these conditions native enzymes breakdown the protein (autolysis), thus affording a substantial amount of purification of chitin. LAB were isolated and identified from various shellfish waste fermentations. Studies on their acid-producing ability revealed a few potentially good strains, identified as Lactobacillus paracasei, Lactobacillus plantarum and Pediococcus sp. The strain of Lactobacillus paracasei was used as a starter culture in the fermentation of shellfish waste in a horizontal rotating bioreactor in order to evaluate the feasibility of the process. The design of the bioreactor was such that it enabled separation of solid and liquid end products during fermentation. Several important fermentation parameters were studied including mode of rotation, concentration of glucose, temperature, rotation rates, loading capacity, type and particle size of waste. Partial purification of the scampi waste was achieved using both batch and fed batch operation, but in the latter, improved purification was achieved at the cost of increased glucose consumption and extended fermentation times. Whilst higher temperatures increased the rates of fermentation, higher rotation rates seemed to have the reverse effect. Mincing the waste helped to increase breakdown of protein whilst larger particles tended to undergo rapid spoilage. Analysis of the chitin product enabled this method to be compared with the conventional method. The results obtained showed that this method is capable of saving large volumes of chemicals and besides producing chitin, the protein liquor by-product could also be used as an ingredient in an animal feed which is not possible by the conventional method.

N-acetil-glucosamina é um composto de importância biotecnológica com grande potencial de aplicação nas áreas de farmácia, medicina e dermatologia. É atualmente produzido pela hidrólise química da quitina, o polissacarideo mais abundante no ambiente marinho e o principal constituinte do exoesqueleto dos artrópodes. No entanto, os processos geralmente utilizados são prejudiciais ao meio ambiente, têm baixo rendimento e alto custo. Este estudo demonstra um dos mais eficientes processos de produção enzimática de N-acetil-glucosamina a partir de -quitina utilizando quitinases bacterianas como uma alternativa sustentável aos processos atuais e isolados de A. caviae com grande potencial para produção de quitinases e derivados de quitina. / N-acetyl-glucosamine (GlcNAc) is a compound of biotechnological importance with great application potential in the areas of pharmacy, medicine and dermatology. GlcNAc is currently produced by the chemical hydrolysis of chitin, the polysaccharide most abundant in the marine environment and the principal constituent of the exoskeleton of arthropods. However, the processes generally used are environmentally unfriendly, have low yield and high cost. This study demonstrates the most efficient processes for enzymatic production of N-acetyl-glucosamine from -chitin using bacterial chitinases as a sustainable alternative to the current processes and A. caviae isolates with great potential for chitinases and chitin derivatives production.

Derivada da quitina, a quitosana apresenta aplicações que variam desde a liberação controlada de fármacos, a filmes de revestimento comestível, biomaterial, lentes de contato. A utilização da beta-quitina para obter a quitosana é vantajosa, pois apresenta-se menos alergênica e mais reativa que a alfa-quitina. A combinação da quitosana com outros materiais pode proporcionar materiais com diferentes propriedades e neste trabalho foram estudadas as misturas de quitosana/gelatina e quitosana/amido, solubilizadas em L-ácido lático. Técnicas como FT-IR, termogravimetria, absorção de água, ensaios de DMA no modo tração e análises reológicas foram usadas em ambas as misturas nas proporções 2:1, 1:1 e 1:2. Os espectros FT-IR mostraram que nos géis e filmes ocorreu interação eletrostática entre os componentes. A termogravimetria mostrou que há diminuição da estabilidade térmica dos componentes quitosana e gelatina quando em presença do L-ácido lático, mas o amido pouco foi afetado pela presença do L-ácido lático. Para os filmes, tanto no caso de quitosana/gelatina quanto no caso quitosana/amido, as misturas mais estáveis termicamente são as que contêm maior quantidade de gelatina ou amido. Isso mostra que é favorável a adição de gelatina ou amido à quitosana quando se deseja maior resistência à temperatura. O aumento da concentração de gelatina ou amido nos filmes de quitosana em L-ácido lático aumentou a absorção de água, o que possibilita a aplicação como suporte na liberação controlada de fármacos. Os ensaios de DMA no modo tração mostraram que o aumento da quantidade de gelatina ou amido nos filmes torna estes mais frágeis. A mistura dos polímeros em diferentes proporções na formação dos géis mostrou que os géis de quitosana/amido adquiriram características de gel em menores frequências que os géis de quitosana/gelatina. O aumento da concentração de gelatina aumenta a temperatura de gelificação, mas para os géis de quitosana/amido nenhum efeito foi observado. Em relação à viscosidade, a gelatina mostrou comportamento newtoniano e as amostras de quitosana e as misturas apresentaram comportamento pseudoplástico, tanto em L-ácido lático como em ácido acético. Os valores de energia de ativação (Ea) de fluxo calculados para quitosana/gelatina e quitosana/amido aumentaram com o conteúdo de quitosana. Os géis de quitosana/amido apresentaram menor Ea que os géis de quitosana/gelatina, apresentando, portanto, maior facilidade para fluir. Os valores de Ea de fluxo para os géis em L-ácido lático são menores que os obtidos para géis em ácido acético. O ácido lático melhorou as propriedades dos géis em relação ao ácido acético, proporcinando filmes mais flexiveís para a liberação controlada de fármacos e géis mais fáceis de deslizar na pele. / Derived from chitin, chitosan has applications ranging from drug delivery system, edible films, biomaterials, contact lenses. The use of beta-chitin for chitosan preparation is advantageous because it is less allergenic and more reactive than alpha-chitin. The association of chitosan with other materials can provide materials with different properties. In this work, mixtures of chitosan/gelatin and chitosan/starch solubilized L-lactic acid were studied. Techniques such as FT-IR, thermogravimetry, water absorption, DMA in traction mode and rheology were studied in both mixtures with proportions of 2:1, 1:1 and 1:2. FTIR spectra showed that both, gels and films, presents electrostatic interaction between the components. The thermogravimetric analysis has shown that there is a decrease in thermal stability of gelatin and chitosan in the presence of L-lactic acid. Thermal stability of starch was little affected by the presence of L-lactic acid. The most thermally stable films are those containing higher amounts of gelatin or starch. This shows that the addition of gelatin or starch to chitosan is favorable. Increasing the gelatin or starch concentration in the films provoked an increase in water absorption, which enables the application to drug delivery system. DMA tests in traction mode showed that increasing the gelatin or starch amount turns films more fragile. Chitosan/starch biomaterial presents gel characteristics at lower frequencies than chitosan/gelatin biomaterial. Increasing the gelatin concentration, an increasing in gelation temperature was observed, however for chitosan/starch gels, no effect was observed. Regarding the viscosity, gelatin showed Newtonian behavior while chitosan samples and the mixtures exhibited pseudoplastic behavior both in L-lactic and acetic acid. The values of flow activation energy calculated for chitosan/gelatin and chitosan/starch increased with chitosan amount. The chitosan/starch gels showed lower energy when compared to chitosan/gelatin gels, thus presenting facility to flow. The values of Ea to chitosan/gelatin and chitosan/starch solubilized in L-lactic acid are smaller than those obtained in acetic acid. L- lactic acid improved the properties of gels with respect to acetic acid, resulting in more flexible films for drug delivery system and gels easier to slide in the skin.

Este trabalho trata da purificação em escala preparativa por técnicas cromatográficas, determinação de seqüência primária parcial, caracterização espectroscópica por dicroísmo circular, fluorescência, infravermelho e investigação de atividades biológicas de duas lectinas ligantes de quitina dos extratos salinos de Artocarpus integrifolia, jaca, e Artocarpus incisa, fruta-pão. Nossos resultados revelaram que as lectinas quitina-ligantes das sementes de jaca e fruta-pão, jackina e frutackina respectivamente, são homólogas entre si, constituindo-se por monômeros de cerca de 14 kDa formados por três subunidades, unidas por pontes S-S. Elas possuem 62% de identidade entre si, são ricas em cisteínas, aminoácidos básicos e serinas e não possuem similares identificadas até o momento, podendo constituir um novo grupo de lectinas na superfamília de lectinas quitina-específicas. Os espectros de dicroísmo circular de jackina e frutackina são similares: ambas são proteínas de estrutura toda-beta, com máximo em torno de 230 nm e mínimo em torno de 214 nm, este último, bastante distorcido por estruturas desordenadas. Os espectros de fluorescência de jackina e frutackina apresentaram máximos de emissão acima de 340 nm, sugerindo que os N-terminais de duas das 3 cadeias de jackina e frutackina (onde os triptofanos estão localizados) estão expostos. Frente a condições extremas de pH e temperatura, monitoradas por CD e fluorescência, observou-se que a estrutura de jackina é vulnerável a pH ácido e termicamente estável. Quanto às atividades biológicas, jackina e frutackina mostraram atividade inibitória de crescimento para Saccharomyces cerevisiae; jackina também mostrou promoção de adesão da linhagem de células de eritroleucemia K562, atividade inibitória para Fusarium moniliforme na concentração de 2,25 mg/mL e atividade hemaglutinante frente a células sangüíneas humanas do sistema ABO e de coelhos, que não foi inibida nem por N-acetilglicosamina, indicando sua preferência por quitina ou seus fragmentos. / This work deals with the preparative-scale purification by chromatographic techniques, the partial primary sequence determination, the spectroscopic characterization by circular dichroism, fluorescence, FT-IR and the investigation of biological activities of two novel chitin-binding lectins from the saline extracts of the seeds of Artocarpus integrifolia, jackfruit, and Artocarpus incisa, breadfruit. Our results revealed that the chitin-binding lectins from jackfruit and breadfruit, jackin and frutackin respectively, are homologous to each other, consiting of monomers of 14 kDa, made up of 3 subunits, linked by S-S bridges. They have 62% of identity between each other; they are rich in cysteines, serines and basic amino acids and they are no homologous to any other known protein, probably constituting a new group of lectins in the chitin-binding lectin superfamily. The CD spectra of jackin and frutackin are similar: both present a beta profile spectra, presenting a maximum about 230 nm and a minimum around 214 nm, this later one, distorted by unordered structures. The fluorescence spectra of jackin and frutackin presented maxima above 340 nm, suggesting that the N-terminals of the 2 up 3 chains of jackin and frutackin (where the tryptophans are) are exposed. Regarding the pH and temperature exposure, monitored by CD and fluorescence, it was observed that the structure of jackin is vulnerable to acid pH and thermally stable. When considered the biological activities, jackin and frutackin presented growth inhibition activity towards Saccharomyces cerevisiae; jackin also promoted the adhesion of the erythroleukemic cell line K562, presented growth inhibition activity towards Fusarium moniliforme at 2,25mg/mL and hemaggluting activity towards rabbit and human red cells from the system ABO, that was not inhibited even by N-acetilglucosamine, suggesting itspreference by oligomers of N-acetilglicosamine or chitin.

A reação de desacetilação da beta-quitina assistida por ultra-som de alta intensidade foi estudada e os efeitos da amplitude e da duração da irradiação e da temperatura sobre a eficiência da reação e as características das quitosanas obtidas foram avaliadas. As espectroscopias de ressonância magnética nuclear e no infravermelho foram empregadas para a caracterização estrutural das amostras, enquanto que a difração de raios X e a microscopia eletrônica de varredura permitiram avaliar as suas características morfológicas. As titulações condutimétricas, as medidas de viscosidade e a cromatografia de exclusão por tamanho foram empregadas para as determinações de grau médio de acetilação e massas molares médias ponderal e viscosimétrica, respectivamente. As quitosanas do Conjunto I foram obtidas nos experimentos em que a amplitude (baixa, intermediária e alta) e a duração (30, 45 e 60 minutos) da irradiação foram variadas, enquanto aquelas do Conjunto II resultaram dos experimentos em que a temperatura (50, 60, 70 e 80ºC) e a amplitude (baixa, intermediária e alta) de irradiação foram variadas, sendo o tempo de irradiação fixado em 30 minutos. Os valores de grau médio de acetilação e a massa molar média viscosimétrica das amostras do Conjunto I variaram nos intervalos 7%<GA<25% e 1,0x105< v M <6,5x105 g/mol, respectivamente. Esses resultados revelam forte correlação entre a amplitude e o tempo de irradiação e as características estruturais das quitosanas, sendo os valores de GA e v M tanto menores quanto maiores a amplitude e a duração da irradiação. O intervalo de variação de GA das amostras do Conjunto II foi muito semelhante àquele das amostras do Conjunto I, porém quitosanas com massas molares mais elevadas foram obtidas no caso do Conjunto II (4,5x105< v M <7,1x105 g/mol). De acordo com os resultados desses experimentos, a temperatura exerce efeito mais importante do que o tempo de irradiação, principalmente sobre o caráter macromolecular das quitosanas obtidas. Independentemente do conjunto considerado (Conjunto I ou Conjunto II), os rendimentos (R) das reações foram elevados e variaram no intervalo 72%<R<90%, entretanto as análises de cromatografia de exclusão por tamanho revelaram que amostras do Conjunto II contêm produtos de baixa massa molar. Os resultados obtidos mostram que a desacetilação da beta-quitina assistida por ultrasom de alta intensidade se constitui em uma nova metodologia mais eficiente do que as convencionalmente empregadas nas indústrias e laboratórios de pesquisa para a produção de quitosanas. Assim, a escolha adequada dos parâmetros do processo permite a produção de quitosanas com massas molares elevadas (5,0-7,0 x 105 g/mol) e baixos graus de acetilação (<10%) em tempos mais curtos (30-60 minutos) e temperaturas mais baixas (<800C) do que as necessárias nos processos convencionais de desacetilação. / The deacetylation of beta-chitin assisted by high intensity ultrasound was studied and the effects of the irradiation amplitude and duration and of the temperature on the reaction efficiency as well as on the chitosan characteristics were evaluated. The nuclear magnetic ressonance and infrared spectroscopies were employed for the structural characterization while the X ray diffraction and the scanning electron microscopy allowed the evaluation of morphological characteristics. The average degree of acetylation was determined by titrimetry while the weight average molecular weight and the viscosity average molecular weight were determined by size-exclusion chromatography and viscometry, respectively. The chitosan samples pertaining to Group I were produced by carrying out the experiments in which the irradiation amplitude (low, intermediate and high) and the time (30, 45 and 60 minutes) were varied, while those of Group II resulted from experiments in which the temperature (50, 60, 70 e 800C) and the irradiation amplitude (low, intermediate and high) were varied, the irradiation time being fixed at 30 minutes. In Group I the values of average degree of acetylation and viscosity average molecular weight of the chitosans ranged as 7%<DA<25% and 1,0x105<Mv<6,5x105g/mol, respectively. These results evidenced a strong relationship between the irradiation amplitude and duration and the structural characteristics of chitosans, the values of DA and Mv being lower the higher the irradiaton amplitude and duration. The chitosans of Group II exhibited a DA range similar to that of the samples of Group I however the molecular weights of the chitosans were higher in Group II (4,5x105<Mv<7,1x105 g/mol). According to the results of such experiments, the temperature effect is more important than the irradiation duration effect, mainly on the macromolecular character of the chitosans. Regardless of the Group (Group I or II), the reaction yields (Y) were high and they were inserted in the range 72%<Y<90%. However, the size-exclusion chromatography analyses showed that the chitosans of Group II contained low molecular weight products. The results of this investigation showed that the high intensity ultrasound-assisted beta-chitin deacetylation is a novel process more efficient than those commonly used to produce chitosan in industries and in research laboratories. Thus, if the parameters process are adequately chosen, chitosans with high molecular weights (5,0-7,0 x 105 g/mol) and low average degrees of acetylation (<10%) are produced in shorter times and at lower temperatures than those tipically of the commonly used processes.

Quitosana é um biopolímero encontrado principalmente na parede celular de crustáceos e é obtida pela desacetilação da quitina. Como biopolímero a quitosana é utilizada como excipiente para medicamentos e composição de alimentos. No entanto a quitosana devidamente purificada para uso farmacêutico ou alimentício tem custo financeiro elevado. Outro fator que contribui para o uso limitado é a falta de procedimento padronizado para desacetilação, o que resulta em materiais com diferentes graus de qualidade, dificultando suas aplicações e controle de qualidade de matéria prima e produto. Este trabalho tem como principal objetivo estabelecer procedimento reprodutível para a extração da quitina e da quitosana, por meio da aplicação dos conceitos de Quality by Design e planejamento de experimentos. A quitosana foi obtida pela desacetilação da quitina de crustáceos pelas etapas de desmineralização, desproteinização e despigmentação. O procedimento técnico para purificação da quitosana foi definido a partir de planejamento fatorial com ponto central para as etapas otimizadas, por meio da aplicação dos conceitos de Quality by Design e planejamento de experimentos. O projeto definiu um procedimento padronizado para purificação da quitosana que pode ser empregado em escala industrial, e financeiramente vantajoso para produção de medicamentos ou alimentos. / Chitosan is a biopolymer found mainly in the cell wall of crustaceans and is obtained by the deacetylation of chitin. As biopolymer chitosan is used as excipient for medicaments and food composition. However, chitosan duly purified for pharmaceutical or food use has a high financial cost. Another factor that contributes to the limited use is the lack of standardized procedure for deacetylation, which results in materials with different grades of quality, hindering their applications and quality control of raw material and product. This work has as main objective to establish a reproducible procedure for the extraction of chitin and chitosan, through the application of the concepts of Quality by Design and planning of experiments. Chitosan was obtained by the deacetylation of chitin from crustaceans through the demineralization, deproteinization and depigmentation stages. The technical procedure for purification of chitosan was defined from a factorial planning with a central point for the optimized steps, through the application of the concepts of Quality by Design and planning of experiments. The project defined a standard procedure for the purification of chitosan that can be used on industrial scale and financially advantageous for the production of medicines or foods.

A quitina é o principal produto obtido do processamento das cascas de crustáceos. Esse biopolímero e o seu derivado, quitosana, têm despertado grande interesse comercial em virtude das possibilidades de aplicações que possuem. O gerenciamento desses resíduos e dos subprodutos gerados nas etapas no processo de obtenção pode ser considerado um modelo de biorrefinaria. A implementação de plantas para extração de quitina e quitosana é um desafio, uma vez que a demanda produtiva deve ser atendida sem causar danos ao meio ambiente. Uma grande variedade de quitosanas com diferentes propriedades físicoquímicas podem ser obtidas variando-se as condições de reação. Essas propriedades dependem da origem da matéria-prima, do seu grau médio de desacetilação, distribuição média dos grupos acetil ao longo da cadeia principal e da sua massa molecular média. Os fornecedores de quitosana comercial geralmente não mencionam a procedência da matéria-prima e pouca ou nenhuma informação é fornecida acerca do seu processamento. Sendo assim, as características e a reatividade do produto final podem variar gerando resultados não reprodutíveis. No presente estudo, foi utilizada a biomassa oriunda de rejeitos da indústria pesqueira de camarão da região de Cananéia SP. As amostras de - quitina foram obtidas por dois procedimentos diferentes: no primeiro, P1, as cascas de camarão após passar pelo pré-tratamento (lavagem, secagem e moagem) foram desproteinizadas para retirada das proteínas em hidróxido de sódio (NaOH) diluído nas concentrações 2%, 5% e 10% e desmineralizadas em ácido clorídrico (HCl) a 20% (v/v) para retirada dos carbonatos; no segundo procedimento, P2, essas etapas foram invertidas. A biomassa resultante foi desacetilada com hidróxido de sódio concentrado a 30%, 40% e 50% em tempos que variaram de 2 a 6 horas. As principais propriedades físico-químicas das amostras de quitosanas obtidas foram determinadas utilizando a espectroscopia na região do infravermelho com transformada de Fourier (FT-IR) para a determinação do grau médio de acetilação, GA, e a técnica de titulação ácido-base mensurada por condutimetria foi utilizada para comparar os resultados; a viscosimetria capilar para a determinação da massa molar média viscosimétrica, Mv , e a difração de raios X (DRX) para avaliar o grau médio de cristalinidade, X. Além disso, foram empregadas as técnicas de microscopia eletrônica de varredura (MEV) para análises morfológicas dos materiais obtidos e a espectrometria de fluorescência de raios X por dispersão de comprimento de onda (WDXRF) para análise química das quitosanas. O GA e o X das amostras diminuíram à medida em que o tratamento se tornou mais vigoroso, enquanto a Mv aumentou. O procedimento 2 foi o mais viável por eliminar a etapa de despigmentação, pois originou amostras com tonalidade mais clara e fáceis de pulverizar. / Chitin is the main product obtained from the processing of crustacean shells. This biopolymer and its derivative, chitosan, have aroused great commercial interest because of the possibilities of applications they have. The management of these wastes and by-products generated in the steps of obtaining processes can be considered a biorefinery model. The implementation of plants for chitin and chitosan extraction is a challenge, since the production demand must be met without causing harm to the environment. A wide variety of chitosan with different physico-chemical properties can be obtained by varying the reaction conditions. These properties depend on the origin of the raw material, its average degree of deacetylation average distribution of the acetyl groups along the backbone and its average molecular weight. Chitosan commercial providers generally do not mention the origin of the raw material and few or no information is provided about the processing. Therefore, the characteristics and reactivity of the final product may vary generating non-reproducible results. The biomass coming from the fishing industry tailings shrimp Cananéia - SP region was used in the present study. Samples of -chitin were obtained by two different procedures: the first, P1, the shrimp shells after passing through the pretreatment (rinsing, drying and grinding) were deproteinized for removal of proteins in diluted sodium hydroxide (NaOH) in concentrations 2%, 5% and 10% and demineralized in hydrochloric acid (HCl) to 20% (v/v) to remove carbonates; in the second procedure, P2, these steps were reversed. The resulting biomass was deacetylated with sodium hydroxide concentrated at 30%, 40% and 50% in times ranging from 2 to 6 hours. The main physicochemical properties of chitosan samples obtained were determined using Fourier transform infrared spectroscopy (FT-IR) to determine the average degree of acetylation, DA, and the acid-base titration technique measured by conductimetry was used to compare the results of chitosan; capillary viscometry to determine the viscosimetric average molecular weight, Mv, and X-ray diffraction (XRD) to evaluate the average degree of crystallinity, X. In addition, scanning electron microscopy (SEM) were employed for morphological analyzes of the obtained materials and wavelength dispersion X-ray fluorescence (WDXRF) for chemical analysis of chitosan. The DA and X of the samples decreased as the treatment became stronger, while Mv increased. Procedure 2 was the most feasible to eliminate the depigmentation step because gave clearer and easier samples spraying.

Derivada da quitina, a quitosana apresenta aplicações que variam desde a liberação controlada de fármacos, a filmes de revestimento comestível, biomaterial, lentes de contato. A utilização da beta-quitina para obter a quitosana é vantajosa, pois apresenta-se menos alergênica e mais reativa que a alfa-quitina. A combinação da quitosana com outros materiais pode proporcionar materiais com diferentes propriedades e neste trabalho foram estudadas as misturas de quitosana/gelatina e quitosana/amido, solubilizadas em L-ácido lático. Técnicas como FT-IR, termogravimetria, absorção de água, ensaios de DMA no modo tração e análises reológicas foram usadas em ambas as misturas nas proporções 2:1, 1:1 e 1:2. Os espectros FT-IR mostraram que nos géis e filmes ocorreu interação eletrostática entre os componentes. A termogravimetria mostrou que há diminuição da estabilidade térmica dos componentes quitosana e gelatina quando em presença do L-ácido lático, mas o amido pouco foi afetado pela presença do L-ácido lático. Para os filmes, tanto no caso de quitosana/gelatina quanto no caso quitosana/amido, as misturas mais estáveis termicamente são as que contêm maior quantidade de gelatina ou amido. Isso mostra que é favorável a adição de gelatina ou amido à quitosana quando se deseja maior resistência à temperatura. O aumento da concentração de gelatina ou amido nos filmes de quitosana em L-ácido lático aumentou a absorção de água, o que possibilita a aplicação como suporte na liberação controlada de fármacos. Os ensaios de DMA no modo tração mostraram que o aumento da quantidade de gelatina ou amido nos filmes torna estes mais frágeis. A mistura dos polímeros em diferentes proporções na formação dos géis mostrou que os géis de quitosana/amido adquiriram características de gel em menores frequências que os géis de quitosana/gelatina. O aumento da concentração de gelatina aumenta a temperatura de gelificação, mas para os géis de quitosana/amido nenhum efeito foi observado. Em relação à viscosidade, a gelatina mostrou comportamento newtoniano e as amostras de quitosana e as misturas apresentaram comportamento pseudoplástico, tanto em L-ácido lático como em ácido acético. Os valores de energia de ativação (Ea) de fluxo calculados para quitosana/gelatina e quitosana/amido aumentaram com o conteúdo de quitosana. Os géis de quitosana/amido apresentaram menor Ea que os géis de quitosana/gelatina, apresentando, portanto, maior facilidade para fluir. Os valores de Ea de fluxo para os géis em L-ácido lático são menores que os obtidos para géis em ácido acético. O ácido lático melhorou as propriedades dos géis em relação ao ácido acético, proporcinando filmes mais flexiveís para a liberação controlada de fármacos e géis mais fáceis de deslizar na pele. / Derived from chitin, chitosan has applications ranging from drug delivery system, edible films, biomaterials, contact lenses. The use of beta-chitin for chitosan preparation is advantageous because it is less allergenic and more reactive than alpha-chitin. The association of chitosan with other materials can provide materials with different properties. In this work, mixtures of chitosan/gelatin and chitosan/starch solubilized L-lactic acid were studied. Techniques such as FT-IR, thermogravimetry, water absorption, DMA in traction mode and rheology were studied in both mixtures with proportions of 2:1, 1:1 and 1:2. FTIR spectra showed that both, gels and films, presents electrostatic interaction between the components. The thermogravimetric analysis has shown that there is a decrease in thermal stability of gelatin and chitosan in the presence of L-lactic acid. Thermal stability of starch was little affected by the presence of L-lactic acid. The most thermally stable films are those containing higher amounts of gelatin or starch. This shows that the addition of gelatin or starch to chitosan is favorable. Increasing the gelatin or starch concentration in the films provoked an increase in water absorption, which enables the application to drug delivery system. DMA tests in traction mode showed that increasing the gelatin or starch amount turns films more fragile. Chitosan/starch biomaterial presents gel characteristics at lower frequencies than chitosan/gelatin biomaterial. Increasing the gelatin concentration, an increasing in gelation temperature was observed, however for chitosan/starch gels, no effect was observed. Regarding the viscosity, gelatin showed Newtonian behavior while chitosan samples and the mixtures exhibited pseudoplastic behavior both in L-lactic and acetic acid. The values of flow activation energy calculated for chitosan/gelatin and chitosan/starch increased with chitosan amount. The chitosan/starch gels showed lower energy when compared to chitosan/gelatin gels, thus presenting facility to flow. The values of Ea to chitosan/gelatin and chitosan/starch solubilized in L-lactic acid are smaller than those obtained in acetic acid. L- lactic acid improved the properties of gels with respect to acetic acid, resulting in more flexible films for drug delivery system and gels easier to slide in the skin.