Desenvolvimento de filmes de quitosana insolúveis em meio ácido com actividade antioxidante

Mestrado em Bioquímica - Bioquímica AlimentarA quitosana é um polissacarídeo constituído maioritariamente por resíduos de D-glucosamina e, em menor número, por resíduos de N-acetil-D-glucosamina com ligação β-1,4. A quitosana tem a capacidade de formar filmes com capacidade antioxidante mas que são solúveis em soluções ácidas. Neste trabalho procedeu-se ao desenvolvimento de uma metodologia para a produção de filmes com base em quitosana insolúveis em meio ácido e com capacidade antioxidante para poderem ser aplicados no processo de vinificação para a substituição da adição de SO2. Com este objectivo foi optimizada uma metodologia para ligação do ácido cafeico (Caf), composto fenólico com actividade antioxidante, por mecanismo radicalar com recurso a agentes oxidantes como persulfato de potássio (KPS) e o hexanitrocerato (IV) de amónio (CAN). A ligação de ácido cafeico aos filmes de quitosana e filmes híbridos de quitosana com (3-cloropropil)trimetoxissilano (CPTMS), com recurso tanto a KPS como a CAN, deu origem a filmes com baixa capacidade antioxidante. No entanto, a ligação de ácido cafeico à quitosana em solução aquosa de ácido acético 5%, utilizando CAN 60 mM, e posterior formação dos filmes, revelou ser a melhor metodologia para a produção de filmes com actividade antioxidante. Estes filmes foram neutralizados com NaOH e reduzidos com NaBH4 com o intuito de aumentar a sua actividade antioxidante. Os filmes de quitosana com ácido cafeico neutralizados apresentaram uma actividade 40% superior em relação aos filmes de quitosana. No entanto, a redução dos filmes não revelou ser vantajosa no incremento da actividade antioxidante. Como se pretendia que os filmes fossem insolúveis em meio ácido, procedeu- se à adição de genipin, um composto natural que tem a função de crosslinker, pois tem a capacidade de ligar simultaneamente duas cadeias de quitosana, evitando que o filme se solubilizasse em meio ácido. A adição do genipin permitiu obter filmes com uma perda de massa de apenas 5%, permitindo uma diminuição da solubilidade em cerca de 75% em relação aos filmes de quitosana com ácido cafeico. Os filmes de quitosana com ácido cafeico e genipin, para além da reduzida solubilidade, apresentaram também actividade antioxidante 80% superior à do filme de quitosana e 54% à do filme de quitosana com ácido cafeico. A actividade antioxidante superior e baixa solubilidade dos filmes de quitosana com ácido cafeico e genipin, produzidos segundo a metodologia desenvolvida neste trabalho, permite que possam ser testados em vinho como possíveis substitutos da adição de SO2.Chitosan is a polysaccharide composed by D-glucosamine and, in a small number, by N-acetyl-D-glucosamine residues in β-1,4 linkages. This polysaccharide has the ability to form films with antioxidant capacity but that are soluble in acid solutions. In this work, a methodology was developed to produce chitosan films with antioxidant activity and insoluble in acid solutions in order to be applied in the vinification process as a substitute of sulfur dioxide With this aim, a methodology was optimized to link caffeic acid, a phenolic compound with antioxidant activity, to chitosan, by a radical mechanism using as oxidant agents, potassium persulfate (KPS) and ammonium hexanitrocerate (IV) (CAN). The linkage of caffeic acid to chitosan and (3- cloropropil)trimethoxysilane (CPTMS) hybrid films using both KPS and CAN gave origin to films with very low antioxidant activity. However, when the reaction of caffeic acid and chitosan was performed in aqueous solution of 5% acetic acid, using CAN 60 mM, the films formed presented antioxidant activity. In order to increase their antioxidant activity, these films were neutralized with NaOH and reduced with NaBH4. The neutralized films prepared with caffeic acid presented 40% higher activity than those prepared only with chitosan. However, the reduction of the films did not increment their antioxidant activity. In order to prepare films insoluble in acid medium, genipin, a natural compound with chitosan crosslinking capacity, was added. The use of genipin allowed to obtain films that only loss 5% of their mass in wine model solutions. When compared with the films of chitosan and caffeic acid, it represents a decrease of 75% in films solubility. These films prepared with chitosan, caffeic acid and genipin presented also an antioxidant capacity 80% higher than that observed for the films prepared only with quitosan and 54% than the films prepared with chitosan and caffeic acid. The higher antioxidant activity and lower solubility in acid solutions of the films prepared with chitosan, caffeic acid and genipin allows their use in wine as possible substitutes of sulfur dioxide

Impact of grape pectic polysaccharides on anthocyanins thermostability

The impact of grape pectic polysaccharides on malvidin-3-O-β-d-glucoside thermostability was evaluated in model solutions. Pectic polysaccharides richer in homogalacturonan domains, with less neutral sidechains (chelator fraction) showed higher binding with malvidin-3-O-β-d-glucoside, by 1H NMR (Ka=505 M-1). Binding affinity with water soluble extract was estimated to be 10-fold lower, possibly due to the presence of neutral branched regions and more compacted structure, hampering the binding. Hydrophobic domains, such as rhamnogalacturonans-I domains in acid soluble polysaccharides, may participate in the formation of complexes with malvidin-3-O-β-d-glucoside. The thermostability of anthocyanin-polysaccharides complexes was evaluated at different temperatures, assessing anthocyanins degradation by HPLC-DAD. Polysaccharides showed to improve anthocyanin thermostability, with chelator and acid extract having the highest impact at lowest temperatures. Electrostatic interactions, additionally stabilized by hydrophobic effect contribute to the anthocyanin-polysaccharides binding and to the consequent thermostabilization. The protection provided by grape pectic polysaccharides foresees innovative anthocyanin food products with improved thermostability and colour features.publishe

Ionic liquid-mediated recovery of carotenoids from the bactris gasipaes fruit waste and their application in food-packaging chitosan films

In this work, a process for the extraction and purification of carotenoids from the fruit Bactris gasipaes was developed. Ethanolic and aqueous solutions of ionic liquids (ILs) and surfactants were evaluated on the extraction of these pigments. Thus, we developed an optimized sustainable downstream process mediated by the best solvent with further isolation of the carotenoids and the recyclability of the IL used. The process was characterized not only in terms of efficiency but also regarding its environmental impact. The recyclability of the solvents as well as the high efficiency (maximum yield of extraction of carotenoids = 88.7 ± 0.9 μgcarotenoids·gdried biomass–1) and the low environmental impact of the integrated process developed in this work were demonstrated. In the end, in order to incorporate functional activity for an alternative food-packaging material, carotenoids were successfully applied on the preparation of chitosan-based films with excellent results regarding their mechanical parameters and antioxidant activity.publishe

The potential of ohmic heating for agar extraction from seaweed

Agar is a polysaccharide with a high commercial value due to its wide range of applications in several industries. Gracilaria is used as the raw material by the agar production companies, being around 54% of the agar commercialized worldwide obtained from this seaweed. Traditional extraction is a process that requires high solvent and energy consumptions and generates large amounts of waste creating the need for greener and more efficient technologies. Ohmic heating consists in a process where an electric current is passed through materials heating them uniformly with a rapid rate. The process has high energy conversion efficiencies, resulting in lower operational costs and in an environmental-friendly system. The ohmic heating can be applied to obtain sub-critical water conditions for extraction of compounds from vegetal tissues, joining the energy and time efficiency with the selective power of sub-critical water. In this context, the aim of this work was to evaluate the effect of ohmic heating on seaweed agar extraction, as an alternative extraction technology. The extraction was performed using a frequency of 25 kHz at 82 °C, during 1 and 2 h, for different proportions of water: ethanol. The extraction yield and sugars composition were identical in all extracts when compared with a conventional extraction, under the same conditions of solvent, temperature, and time. However, the gel strength of agar was significantly higher for the extracts obtained with ohmic treatment in water. These results show the potential of ohmic technology as a low cost and an environmental-friendly alternative for agar extraction from seaweed.The authors acknowledge to FCT/MEC for the financial support of the project “OH2O” (POCI-01-0145-FEDER-029145) and Research Units CICECO (UID/CTM/50011/2019) and QOPNA (UID/QUI/00062/2019), through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. This work was also funded by national funds (OE), through FCT – Fundação para a Ciência e a Tecnologia , I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.”info:eu-repo/semantics/publishedVersio

Chitosan/fucoidan multilayer nanocapsules as a vehicle for controlled release of bioactive compounds

Abstract Hollow multilayer nanocapsules were successfully prepared through layer-by-layer assembly of two bioactive polysaccharides, chitosan and fucoidan. The stepwise adsorption of 10 chitosan/fucoidan layers and the consequent formation of a multilayer film on polystyrene nanoparticles (used as templates) were followed through ζ-potential measurement and the removal of the polystyrene core was confirmed by FTIR analysis. The chitosan/fucoidan nanocapsules morphology and size were evaluated by SEM and TEM, that showed that after the core removal, the nanocapsules maintained their spherical shape and a decrease of size occurred. A cationic bioactive compound, poly-L-lysine (PLL), was chosen to evaluate the loading and release behaviour of the nanocapsules. The chitosan/fucoidan nanocapsules showed a good capacity for the encapsulation and loading of PLL, which shows to be influenced by the initial PLL concentration and the method of encapsulation used. The results of fitting the linear superimposition model to the experimental data of PLL release suggest an anomalous behaviour, with one main polymer relaxation. The PLL release was found to be pH-dependent: at pH 2 relaxation is the governing phenomenon and at pH 7 Fick's diffusion is the main mechanism of PLL release. Chitosan/fucoidan nanocapsules is a promising delivery system for water soluble bioactive compounds, such as PLL, showing a great potential of application in food and pharmaceutical industriesThe authors Ana I. Bourbon, Miguel A. Cerqueira, Elia Maricato, and Claudia Nunes are recipient of fellowships from the Fundacao para a Ciencia e Tecnologia, POPH-QREN and FSE (FCT, Portugal) through grants, SFRH/BD/73178/2010, SFRH/BPD/72753/2010, SFRH/BD/87245/2012 and SFRH/BPD/46584/2008, respectively. The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Also, thanks are due to Fundacao para a Ciencia e a Tecnologia (FCT, Portugal), European Union, QREN, FEDER and COMPETE for funding the QOPNA research unit (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296). The authors would like to acknowledge to Rui Fernandes from IBMC, University of Porto for assistance in TEM analysis and to Edith Ariza from SEMAT/UM by the support in SEM analysis

Fractionation of Isochrysis galbana Proteins, Arabinans, and Glucans Using Ionic-Liquid-Based Aqueous Biphasic Systems

Isochrysis galbana (I. galbana) is a microalgae species rich in biomolecules of high commercial value and industrial relevance. Despite the numerous studies done on the extraction of different compounds from microalgae, the complete valorization of the biomass under the biorefinery concept is scarce or even inexistent. The mixture of different compounds found in the different extracts obtained from the microalgae biomass is one of the most important drawbacks in the field. Despite the large interest of academia and industry in the different classes of bioactive compounds composing microalgae, these are still poorly explored due to the low efficiency of the downstream processes used up to date. In this context, this work proposes the development of an efficient purification process by applying ionic liquid (IL)-based aqueous biphasic systems (ABS) to separate proteins from arabinose- and glucose-rich polysaccharides. For this purpose, the nature of the inorganic salt, the IL structural features (anion and alkyl chain length) and the extraction point (using different inorganic salt concentrations) were the conditions optimized. After the proper selection of the most performant IL-ABS (%EEcarb = 71.21 ± 5.21% to the bottom phase and %EEprot = 100% to the top phase), a complete downstream process was developed and implemented, in which the isolation of the target biomolecules (proteins, arabinose- and glucose-rich polysaccharides) and the reuse of the phase formers and main solvents applied was considered. In summary, the approach proposed using IL-ABS appears as a simple and efficient method of purification easily integrated into continuous flow processes, thus demonstrating its industrial potential.publishe

Chemical Composition and Antioxidant Potential of Five Algae Cultivated in Fully Controlled Closed Systems

In this study, the chemical composition and antioxidant profile of five edible macroalgae, Fucus vesiculosus, Palmaria palmata, Porphyra dioica, Ulva rigida, and Gracilaria gracilis, cultivated in fully controlled closed systems, were determined. Protein, carbohydrates, and fat contents ranged between 12.4% and 41.8%, 27.6% and 42.0%, and 0.1% and 3.4%, respectively. The tested seaweeds presented considerable amounts of Ca, Mg, K, Mn, and Fe, which reinforce their favorable nutritional profile. Regarding their polysaccharide composition, Gracilaria gracilis and Porphyra dioica were rich in sugars common to agar-producing red algae, and Fucus vesiculosus was composed mainly of uronic acids, mannose, and fucose, characteristic of alginate and fucoidans, whereas rhamnose and uronic acid, characteristic of ulvans, predominated in Ulva rigida. Comparatively, the brown F. vesiculosus clearly stood out, presenting a high polysaccharide content rich in fucoidans, and higher total phenolic content and antioxidant scavenging activity, determined by DPPH and ABTS. The remarkable potential of these marine macroalgae makes them excellent ingredients for a wide range of health, food, and industrial applications

Chitosan-genipin film, a sustainable methodology for wine preservation

A novel approach is described using chitosan–genipin films as a sustainable method for wine preservation. Films prepared from fungi and from shrimp by-products showed similar mechanical and chemical properties. In addition, shrimp derived films showed no IgE positive reaction against shellfish allergenic compounds. The chitosan–genipin films were used to produce white wines without the addition of sulphur dioxide as a preservative. These wines showed lower susceptibility to browning, with organoleptic characteristics comparable to those prepared using sulphur dioxide. It is shown that the formation of iron-tartrate–chitosan complexes promotes the decrease of iron and other metal availability, minimizing oxidation reactions, as well as inhibiting microbial growth. The overall volatile character of the wines was maintained. However, the presence of chitosan–genipin films promotes the occurrence of Maillard and Strecker reactions, producing volatile compounds with positive wine aroma scents. The use of chitosan–genipin films in winemaking is an innovative, green and environmentally friendly technology that could be easily implemented at an industrial scale with no additional costs