Ana Cristina Braga Pinheiro

Universidade do Minho Escola de Engenharia Ana Cristina Braga Pinheiro behaviour in vitro heir Development of nanostructures for tructures for application in food application in food aluation of t v technology – evaluation of their in vitro behaviour velopment of nanos De technology – e o aga Pinheir Ana Cristina Br 3 1 UMinho|20 August, 2013 Universidade do Minho Escola de Engenharia Ana Cristina Braga Pinheiro Development of nanostructures for application in food technology – evaluation of their in vitro behaviour Dissertation for PhD degree in Chemical and Biological Engineering Supervisors of the thesis Professor António Augusto Vicente Professor Manuel A. Coimbra August, 2013 AGRADECIMENTOS Durante este percurso muitos foram os que me ajudaram e contribuíram para que eu chegasse ao fim desta etapa e aos quais não poderia deixar de agradecer. Em primeiro lugar, os meus agradecimentos vão para o meu orientador, Professor António Vicente, pela confiança que depositou em mim, por todo o apoio, disponibilidade e amizade. As suas excepcionais qualidades científicas e humanas foram sem dúvida determinantes para a concretização deste trabalho. De igual modo agradeço ao meu co-orientador, Professor Manuel Coimbra, por todo o apoio científico, pela confiança que depositou em mim e pela disponibilidade demonstrada. À instituição de acolhimento, Centro de Engenharia Biológica da Universidade do Minho pela disponibilização do espaço e equipamento necessários à realização deste trabalho. Agradeço ao Sr. Santos e à Engª Madalena Vieira o apoio técnico e toda a disponibilidade demonstrada sempre que necessário. Aos meus colegas e amigos do LIP agradeço o espírito de entreajuda, a partilha de ideias, a constante boa disposição e o excelente ambiente de trabalho que me proporcionaram! Agradeço em especial à Isa, ao Miguel e ao Hélder pela colaboração activa neste trabalho. Sem dúvida que pertencer a uma equipa tão dinâmica tornou a realização deste trabalho uma tarefa muito mais fácil! Gostaria também de expressar uma palavra de agradecimento à Mafalda Quintas e à Professora Graça que muito contribuíram para o desenvolvimento deste trabalho. Aos colegas do departamento de química da Universidade de Aveiro, em especial à Cláudia e à Élia, agradeço a ajuda na análise dos polissacarídeos. I would also like to express my gratitude to Dr. Mike Boland for giving me opportunity to do part of my work at Riddet Institute in New Zealand and to Dr. Mita Lad for being so supportive and helpful! I am thankful to all the staff and research fellows in Riddet Institute for receiving so well, especially to Amit and Janiene for the technical support and to Natascha, Ajit and Noelia for making my passage through the beautiful New Zealand so much pleasant! Um obrigado muito especial ao Bruno pelo amor, companheirismo e por me incentivar a ser sempre melhor! Obrigada por estares sempre presente! Finalmente agradeço à minha Família, especialmente aos meus pais e irmã, a quem devo tudo o que sou, pelo incentivo e apoio que sempre me deram! iii This thesis was financially supported by a PhD scholarship from Fundação para a Ciência e Tecnologia (Ref.: SFRH/BD/48120/2008), inserted in the Programa Potencial Humano Quadro de Referência Estratégico Nacional (POPH - QREN) Tipologia 4.1 – Formação Avançada. The POPH-QREN is co-financed by Fundo Social Europeu (FSE) and by Ministério da Ciência, Tecnologia e Ensino Superior (MCTES). iv ABSTRACT The emerging field of nanotechnology offers new challenges to the food industry either by offering novel tools for the development of strategies to improve food quality and human health, or by the introduction of questions about the behaviour of nanostructures within the human body. Nanotechnology holds a great potential to generate very innovative solutions and to provide food technologists and manufacturers with instruments to meet the ever- growing consumer demands in very diverse aspects related with the foods they eat: safety, quality, health-promotion and novelty. However, the application of nanostructures to foods is hindered by very pertinent problems, which could be summarized in two issues: edibility (only edible materials must be used for their production) and functionality/behaviour once inside the human body, that is raising safety concerns among the consumers, and therefore demands an evaluation (ideally) in vivo, or at least in vitro. In this context, the two main challenges addressed in this thesis were to develop stable nanostructures for food applications and to evaluate their in vitro behaviour. The strategy adopted included the development and characterization of edible nanostructures, incorporation of bioactive compounds and evaluation of their behaviour when subjected to digestion in artificial gastrointestinal (GI) systems. In particular, the research undertaken was based on three different nanostructures: nanofilms composed of κ-carrageenan and chitosan, curcumin nanoemulsions stabilized by different emulsifiers and multilayer nanocapsules composed of chitosan and fucoidan. The interactions between κ-carrageenan and chitosan in nanofilms have been investigated and the results showed that κ-carrageenan/chitosan interaction is an exothermic process and that the alternate deposition of κ-carrageenan and chitosan results in the formation of a stable multilayer structure mainly due to the electrostatic interactions existing between the two polyelectrolytes. The κ-carrageenan/chitosan nanolayers exhibit good gas barrier properties and therefore offer great potential to be used e.g. to coat food systems. Also, the model cationic compound methylene blue (MB) was incorporated in different positions of the κ-carrageenan/chitosan nanolayered coating and its loading and in vitro release behaviour were evaluated. Loading results suggest that MB was able to diffuse into the κ-carrageenan/chitosan nanolayered coating and not only adhered to the surface of the layer immediately below it. For most of the tested conditions, MB release from the κ- carrageenan/chitosan nanolayered coatings was successfully described by the linear superimposition model, which allowed concluding that MB transport is due to both v concentration gradient and polymer relaxation in the nanolayers. However, depending on temperature and pH of the medium and on the position of MB incorporated on the nanolayered coatings, different mechanisms prevailed. These results lead to conclude that the κ-carrageenan/chitosan nanolayered coatings represent a promising platform from which the controlled release of different bioactive compounds may be explored. Regarding curcumin nanoemulsions, they were prepared using four different emulsifiers: Tween 20 (non-ionic), Sodium Dodecyl Sulphate (SDS, anionic), Dodecyltrimethylammonium Bromide (DTAB, cationic) and lactoferrin (protein). Their formulation was optimized and their stability was evaluated during 90 days. DTAB-stabilized emulsions revealed to be the least stable, showing a phase separation, the highest colour change and the highest decrease in the magnitude of ζ-potential. Nanoemulsions stabilized by lactoferrin revealed to be at least as stable as nanoemulsions stabilized by synthetic emulsifiers, thus showing the great potential of lactoferrin to substitute synthetic emulsifiers, which is in line with the current trend of food industry. A human gastric simulator was used as in vitro digestion model (in which stomach, duodenum, jejunum and ileum steps were performed) to evaluate the impact of emulsifier (Tween 20, SDS and DTAB) charge on the digestion of curcumin nanoemulsions. The emulsifier charge had a significant effect on droplet size, particle electric charge and microstructure of curcumin nanoemulsions during the simulated digestion, which consequently influenced free fatty acids release and curcumin bioavailability. Results showed the positively charged DTAB-stabilized emulsions to be the least stable during the digestion process, exhibiting the largest increase in droplet size and eventual phase separation. This also contributed to the observed low bioavailability of curcumin. Conversely, emulsions stabilized with Tween 20 showed retention of emulsion structure (high surface area) and greater free fatty acid production, which could explain increased curcumin bioavailability. A dynamic digestion model, comprising the simulation of stomach, duodenum, jejunum and ileum, was developed and used to evaluate the impact of alginate coating on the digestion of curcumin nanoemulsions stabilized by lactoferrin. Results suggested that alginate coating may be able to control the rate of lipid digestion and free fatty acids adsorption within the GI tract, but encapsulated lipids are digested to the same extent, releasing the lipophilic bioactive compound. Biodegradable hollow nanocapsules were developed through alternate deposition of 10 chitosan/fucoidan layers on polystyrene (PS) nanoparticles, used as templates, followed by vi removal of the PS core. Poly-L-lysine (PLL) was entrapped in the nanocapsules and its loading and release behaviour was evaluated. Chitosan/fucoidan nanocapsules showed a good capacity for the encapsulation and loading of PLL and the results of fitting the linear superimposition model to the experimental data of PLL release suggested an anomalous release behaviour, with one main polymer relaxation and PLL release was found to be pH- dependent. Due to their bioactive and non-toxic nature and to their responsiveness at different pHs, chitosan/fucoidan hollow nanocapsules showed

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