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Previous issue date: 2015-10-08 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A encapsulação de moléculas bioativas é utilizada há décadas pelas industrias de alimentos e representa uma real oportunidade de desenvolvimento de produtos inovadores. Dada a sua versatilidade funcional, as proteínas do leite, em particular as proteínas do soro de leite, tem sido utilizadas para fins de encapsulação por meio de diferentes técnicas. Complementarmente, estudos recentes mostraram a habilidade de proteínas alimentares de carga oposta de se co-associar formando micro-esferas através da coacervação complexa. Compreender as forças que governam o processo de coacervação de hetero-proteínas e o efeito da presença de pequenos ligantes (bioativos) são pré-requisitos para o uso de coacervados complexos de hetero-proteínas como agentes de encapsulação. Neste contexto, o objetivo do meu projeto de tese foi entender o mecanismo de coacervação complexa entre β-lactoglobulina (β-LG) e lactoferrina (LF) na ausência ou na presença de pequenos ligantes. As condições ótimas para a coacervação entre β-LG e LF foram identificadas como sendo entre os pH 5.4 – 6.0 e em presença de um excesso molar de β-LG. Interessantemente, LF demonstrou uma seletividade de coacervação com a β-LG A, a isoforma ligeiramente mais eletronegativa. A nivel molecular, a presença de dois sítios de interação da β-LG com a LF foram evidenciados. Em complemento, hetero-complexos como o pentâmero LF(β-LG 2 ) 2 e outros complexos maiores (LFβ- LG 2 ) n foram identificados como constituintes da fase coacervada. Para avaliar o efeito da presença de pequenos ligantes na coacervação complexa entre β-LG e LF, foram usados modelos de moléculas hidrofóbica (ANS) e hidrofílica (ácido fólico). Embora nas condições experimentais os pequenos ligantes não tenham interagido com a β-LG, ambos interagiram com a LF induzindo sua auto-associação em nano- partículas. Concentrações relativamente elevadas de pequenos ligantes afetaram a interação entre as duas proteínas levando a uma transição entre os regimes de coacervação e agregação. / Le bénéfice de l’encapsulation des molécules bioactives a séduit les industries agroalimentaires depuis plusieurs décennies et constitue toujours un levier de développement pour des produits innovants. Plus récemment des études ont montré la capacité de protéines alimentaires de charge opposée à s’assembler en microsphères par coacervation complexe. La compréhension des forces gouvernant le processus de coacervation complexe entre protéines et l’influence exercée par la présence de petits ligands (bioactifs) demeurent des prérequis pour l’utilisation des coacervats complexes de protéines comme agent d’encapsulation. Dans ce contexte, l’objectif de mon projet de thèse a été de comprendre le mécanisme de coacervation complexe entre une protéine chargée négativement, la β-lactoglobuline (β-LG), et une protéine chargée positivement, la lactoferrine (LF), issues du lactosérum en absence et en présence de petits ligands. Les conditions optimales de coacervation entre la β-LG et la LF ont été définies entre pH 5.4 et 6.0 ainsi qu’en présence d’un excès de β-LG. La LF a présenté une coacervation préférentielle avec le variant A de la β-LG qui se distingue du variant B par la substitution de 2 acides aminés. Au niveau moléculaire, deux sites de fixation de la β-LG sur la LF ont été identifiés. En outre, par la mesure d’une part des coefficients de diffusion rotationnel et d’autre part de la cinétique de diffusion des entités moléculaires constituant les coacervats, il est suggéré que ces derniers sont formés à partir de β-LG libre, de pentamère, LF(β- LG 2 ) 2 , ainsi que des entités plus larges, (LFβ-LG 2 ) n . Afin d’évaluer l’effet de la présence de petits ligands sur la coacervation complexe entre la β-LG et la LF, des ligands modèles, l’un hydrophobe (ANS), l’autre hydrophile (acide folique) ont été utilisés. Dans les conditions expérimentales testées ces deux ligands n’ont pas d’affinité pour la β-LG, mais après interaction avec la LF ils sont capables d’induire son auto-association en nanoparticules. En concentrations élevées de ligands, la coacervation complexe entre la β-LG et la LF est perturbée et une transition vers un régime d’agrégation est observée. / Encapsulation of bioactives has been used by the food industries for decades and represents a great potential for the development of innovative products. Given their versatile functional properties, milk proteins in particular from whey have been used for encapsulation purposes using several encapsulation techniques. In parallel, recent studies showed the ability of oppositely charged food proteins to co-assemble into microspheres through complex coacervation. Understanding the driving forces governing heteroprotein coacervation process and how it is affected by the presence of ligands (bioactives) is a prerequisite to use heteroprotein coacervates as encapsulation device. In this context, the objective of my thesis work was to understand the mechanism of complex coacervation between β-lactoglobulin (β-LG) and lactoferrin (LF) in the absence and presence of small ligands. The conditions of optimal β-LG - LF coacervation were found at pH range 5.4-6 with a molar excess of β-LG. Remarkably, LF showed selective coacervation with β-LG A, the slightly more negative isoform. At molecular level, the presence of two binding sites on LF for β-LG was evidenced. Moreover, the heterocomplexes such as pentamers LF(β-LG 2 ) 2 and quite large complexes (LFβ-LG 2 )n were identified as the constituent molecular species of the coacervate phase. To evaluate the β-LG - LF complex coacervation in the presence of small ligands, models of hydrophobic (ANS) and hydrophilic molecules (folic acid) were used. Although under the experimental conditions tested the small ligands did not interact with β-LG, both interacted with LF inducing its self- association into nanoparticles. High relative concentrations of small ligands affected the interaction between the two proteins leading to a transition from coacervation to aggregation regime.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:123456789/7801 |
| Date | 08 October 2015 |
| Creators | Tavares, Guilherme Miranda |
| Contributors | Bouhallab, Saïd, Croguennec, Thomas, Basílio, Eduardo, Perrone, Ítalo Tuler, Carvalho, Antônio Fernandes de |
| Publisher | Universidade Federal de Viçosa |
| Source Sets | IBICT Brazilian ETDs |
| Language | English |
| Detected Language | French |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFV, instname:Universidade Federal de Viçosa, instacron:UFV |
| Rights | info:eu-repo/semantics/openAccess |
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