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- Chitosan mouthwash: toxicity and in vivo validationPublication . Costa, E. M.; Silva, S.; Costa, M. R.; Pereira, M.; Campos, D. A.; Odila, J.; Madureira, A. R.; Cardelle-Cobas, A.; Tavaria, F. K.; Rodrigues, A. S.; Pintado, M. M.A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashesas it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes.As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, thebiological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT andV79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. Theresults showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercialmouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca.5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwashpossessed significantly higher antimicrobial activity. The conjunction of these results proves that thechitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.
- Enzymatic soy protein hydrolysis: a tool for biofunctional food ingredient productionPublication . Coscueta, Ezequiel R.; Campos, Débora A.; Osório, Hugo; Nerli, Bibiana B.; Pintado, ManuelaThis work aimed to evaluate the digestive stability of the peptides previously identified from a Corolase PP soy protein hydrolysate (SPH) and to respond to the uncertainty about the merit of controlled hydrolysis. For this purpose, we applied an empirical and theoretical analysis, determining peptide sequences, oxygen radical scavenging (ORAC) and ACE inhibitory (iACE) activities, and the effect of hydrolysis on solubility. Results showed that during digestion most of SPH peptides were degraded as smaller ones. However, both SPH bioactivities improved significantly after digestion (3.9 ± 0.1 μmol TE/mg protein for ORAC and IC50 = 52 ± 4μg protein/mL for iACE) with similar values for soy protein isolate (SPI). With respect to solubility, the controlled hydrolysis considerably increased this functional property. In conclusion, the results indicated that controlled enzymatic hydrolysis of SPI with Corolase PP produced an ingredient more apt to be incorporated in certain nutritional or nutraceutical formulations.
- Management of fruit industrial by-products - a case study on circular economy approachPublication . Campos, Débora; Gómez-García, Ricardo; Vilas-Boas, Ana A.; Madureira, Ana Raquel; Pintado, Maria ManuelaThe management of industrial fruit by-products is important not only to decrease the volume of food waste accumulated in the landfills but also to develop strategies through reuse with the purpose to valorise and add economic value. The disposal of food waste leads to di erent global issues in di erent sectors, such as social, environmental and economical. These by-products represent a rich source of valuable compounds (polyphenols) with high antioxidant activity, which can be extracted through biotechnological methodologies for future industrial applications. In this context, the management of fruit by-products is challenged to move from a linear economy to a circular economy. Therefore, the purpose of this review is to provide a critical view of an integrated valorisation of fruit by-products to overcome a global issue, via the production of antioxidant extracts with high economic value. A case study of pineapple processing industrialization in a circular economy is explored and discussed.
- Platform design for extraction and isolation of Bromelain: complex formation and precipitation with carrageenanPublication . Campos, Débora A.; Valetti, Nadia Woitovich; Oliveira, Ana; Pastrana-Castro, Lorenzo M.; Teixeira, José A.; Pintado, Maria Manuela; Picó, GuillermoThe main objective of this work was to investigate for the first time the molecular mechanism of com-plex formation between bromelain (a positively charged enzyme) and carrageenan (a natural strongpolyelectrolyte, negatively charged) using spectroscopy techniques and thermodynamic approaches. TheBromelain-Carrageenan complex showed a maximal non-solubility at pH around 5.1. The solubility wasdependent on pH and ionic strength of the medium. To re-dissolve the formed complex, the pH waschanged and 500 mM of NaCl was added to the initial solution, proving the columbic mechanism forthe formation of non-soluble complex. The formation of the carrageenan-bromelain complex increasedin 8◦C the enzyme thermal stability, while its biological activity was not modified. The amount of totalenzyme recovered in solution after precipitation with around 0.08% w/v of carrageenan was 85–90%.
- Study of antimicrobial activity and atomic force microscopy imaging of the action mechanism of cashew tree gumPublication . Campos, Débora A.; Ribeiro, Alexandra C.; Costa, Eduardo M.; Fernandes, João C.; Tavaria, Freni K.; Araruna, Felipe Bastos; Eiras, Carla; Eaton, Peter; Leite, José Roberto S. A.; Pintado, M. ManuelaThe aim of this work was to evaluate the antimicrobial potential of two grades of cashew tree gum (crude and purified) against eight microorganisms and to analyze the mechanism of cashew tree gum antimicrobial action via atomic force microscopy (AFM) imaging. The results indicated strong antimicrobial properties of pure cashew tree gum against all tested microorganisms, except for Candida albicans and Lactobacillus acidophilus. On the other hand crude cashew gum showed antimicrobial activity only against Gram-positive bacteria (MRSA, MSSA, Listeria innocua and Enterococcus faecium). Atomic force microscopy imaging showed that pure cashew tree gum lead to bacterial cell collapse. In conclusion cashew tree gum presented relevant antimicrobial activity against most of the studied bacteria, and the purification of the cashew gum affected its antimicrobial spectrum.
- Technological stability of solid lipid nanoparticles loaded with phenolic compounds: drying process and stability along storagePublication . Campos, Débora A.; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Maria Manuela; Gomes, Ana MariaSolid lipid nanoparticles (SLNs) have been widely studied and tested as vehicles for natural compounds. Recently, Witepsol and Carnauba SLNs were shown to be effective systems for the entrapment of rosmarinic acid (RA) and herbal extracts. In the present work, the improvement of stability and bioactivity of these systems was studied. Thus, the freeze-drying of SLNs produced with Witepsol and Carnauba waxes loaded with RA and herbal extracts (sage and savory) were tested. The use of three different cryoprotectants (glucose, mannitol and trehalose) at two different concentrations (5 and 10%, w/v) were evaluated. Furthermore, the prepared SLNs were stored under different conditions (atmosphere, temperature, absence or presence of light) and in different packaging materials, over 365 days. The effect on the SLNs physical stability and bioactivity was assessed. The most suitable cryoprotectant was mannitol at 10% (w/v) for all formulations tested. The solid state of SLNs, with storage at room temperature, in glass flasks, protected from light and under N2 controlled atmosphere were the best storage conditions in which the SLNs bioactivity was maintained during 365 days.
- Development and characterization of functional ingredients through valorization of pineapple by-products : production, bioactivity and potential applicationPublication . Campos, Débora Andreia Campelo; Pintado, Maria Manuela Estevez; Pastrana, LorenzoAs indústrias de processamento de frutas produzem toneladas diárias de resíduos, sendo os subprodutos de abacaxi um dos mais importantes, devido ao tamanho do fruto e, devido ao fato de 60% do peso do seu peso ser descartado como resíduo. A valorização total de subprodutos de abacaxi, por meio da química verde e de tecnologias sustentáveis, visando a obtenção de zero resíduos, é uma das possíveis abordagens para realizar uma valorização integrada destes subprodutos através de uma abordagem de biorrefinaria. O objetivo principal deste trabalho de doutoramento consistiu em utilizar subprodutos do abacaxi (cascas e centros) no desenvolvimento de ingredientes funcionais, desde concentrados de enzimas, extratos prebióticos/ antioxidantes e farinhas funcionais, e caracterizar e validar as atividades biológicas e as capacidades tecnológicas desses novos ingredientes. Primeiro, os centros e cascas do abacaxi foram fracionados gerando dois sumos (frações líquidas) e duas frações sólidas. As frações líquidas do centro representam 5% e da casca 17% do peso total do abacaxi. Ambas as frações líquidas apresentaram alto teor de açúcares simples, representando ca. 65% no caule e ca. de 67% na casca; ambas frações contêm menos de 1% de fibra dietética solúvel. Além disso, foi encontrado alto conteúdo de compostos fenólicos totais ca. 1.3% para a fração líquida do caule e ca. 0.7% para a fração líquida da casca, com elevador teor de vitamina C, associada também a uma elevada atividade antioxidante medida pelos métodos de ABTS e ORAC. Por outro lado, a fração sólida do caule representa ca. 44%, enquanto que a fração sólida da casca representa ca. 38% de abacaxi total, contendo proporções muito diferentes de hidratos de carbono. A fração sólida do caule contém 23% de açúcares simples, 5% de fibra dietética solúvel e 45% de fibra dietética insolúvel, enquanto que fração sólida da casca contém 28% de açúcares simples, 8% de fibra dietética solúvel e 38% de fibra dietética insolúvel, demonstrando um grande potencial como ingrediente rico em fibra. A composição dos hidratos de carbono estruturais mostrou diferenças, principalmente no teor total de celulose e lignina insolúvel. Uma precipitação com polissacáridos foi aplicada às frações líquidas do abacaxi para extrair a principal enzima presente - a bromelaína, uma importante enzima natural que é amplamente aplicada na indústria. A metodologia de separação desenvolvida baseada na precipitação com carragenana, representa uma alternativa natural às metodologias tradicionais de separação. Uma otimização experimental foi aplicada aos subprodutos de abacaxi para definir os valores ótimos responsáveis pela separação da bromelaína. Alto rendimento de recuperação (cerca dos 80%) de bromelaína ativa foi obtida para ambos os sumos, sendo possível extrair 0.26 g de bromelaína a partir de 100 g de subprodutos de abacaxi usando baixas concentrações de polissacarídeo (0.2-0.3%, p/v). Ambas as frações líquidas foram submetidas a um sistema gastrointestinal (TGI) simulado para compreender o impacto na composição dos bioativos presentes. Observou-se um elevado grau de hidratos de carbono digeridos, uma vez que os hidratos carbono de alto peso molecular, após a digestão bucal e duodenal, transformaram-se em moléculas de menor tamanho (tri-, di- e monossacarídeos). O conteúdo total de polifenóis aumentou ao longo do TGI, assim como a atividade antioxidante, medida por ABTS e ORAC, devido à libertação de polifenóis complexados com outras moléculas. A análise de composição de polifenóis mostrou que o ácido clorogénico está em maior concentração, combinado com outros como os ácidos cafeíco, cumárico e ferúlico, principalmente libertados após a digestão com TGI. A fração líquida do caule foi selecionada para estudar o potencial prebiótico, revelando elevada atividade em cinco estirpes probióticas (duas de lactobacilos e três de bifidobactérias). A atividade prebiótica foi medida após cada compartimento do TGI, a qual diminuiu ligeiramente ao longo do sistema. No entanto, a fração residual evidenciou capacidade para modular o crescimento e o metabolismo das estirpes probióticas. As frações sólidas também foram submetidas à simulação do TGI e a fração remanescente foi fermentada in vitro num modelo de fezes humanas. Ambas as frações evidenciaram elevadas concentrações de ácidos ferúlico e cumárico (cascas: 70 mg 100 g-1 e 113 mg 100 g-1 e caules: 95 mg 100 g-1 e 68 mg 100 g-1, respetivamente) que foram detetados após a simulação do TGI, bem como após 48 h de fermentação com microbiota intestinal, demonstrando ter um alto potencial antioxidante durante todas as fases do TGI. A fração sólida do caule promoveu o crescimento de Lactobacillus spp. e Bifidobacterium spp., enquanto a fração sólida da casca promoveu principalmente o crescimento de Lactobacillus spp. O crescimento desses grupos levou à produção de ácidos gordos de cadeia curta (AGCC), principalmente ácido propiónico e acético, que modularam positivamente o metabolismo intestinal. A prova de conceito de um dos ingredientes desenvolvidos foi realizada através da aplicação de bromelaína natural em um processo de amaciamento de um fiambre tradicional. A concentração mais baixa de bromelina (1 mg mL-1) permitiu o amaciamento da carne de porco. Através de um desenho experimental fatorial, os intervalos de valores e as condições ótimas de aplicação de enzima foram selecionadas: 1 mg de bromelaína por mL de salmoura. Os resultados deste trabalho experimental contribuirão para a sustentabilidade das indústrias de processamento de frutas num contexto de economia circular, através do desenvolvimento de ingredientes de valor agregado com impacto bioativo e funcional. Além disso, o desenvolvimento de seis novos ingredientes funcionais, através da aplicação de um processo a jusante integrado com obtenção de resíduos ZERO, ajudará as indústrias de processamento de frutas a implementar soluções de baixo custo, com produtos finais de alto valor agregado, o que permite a reintegração do valor económico, com a geração de novos fluxos de receita e o incentivo a uma sociedade mais sustentada.
- Polyphenol extraction by different techniques for valorisation of non-compliant portuguese sweet cherries towards a novel antioxidant extractPublication . Vilas-Boas, Ana A.; Campos, Débora A.; Nunes, Catarina; Ribeiro, Sónia; Nunes, João; Oliveira, Ana; Pintado, ManuelaCurrently, there is special interest in the recovery of polyphenols from non-compliant fruits that have no market value; e orts to find value-added solutions for these food areas are a key option for a sustainable bio-economy. Saco cherries are a traditional Portuguese cherry variety, and although they are a nutritionally important food, rich in powerful dietary polyphenols, significant amounts of these cherries are not sold due to their small size. In this context, this work aimed to select the best method to produce novel antioxidant polyphenol-rich extracts from low calibre and non-compliant Saco cherries. Based on the results, microwaves-assisted extraction (MAE) allowed us to obtain a polyphenol-rich extract with a high antioxidant capacity (50.46 + 1.58 mg Trolox equivalent (TE)/g dry extract (DE) by oxygen radical absorbance capacity (ORAC), 10.88 + 0.38 mg ascorbic acid equivalent (AA)/g DE by 2-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and 9.58 + 0.42 mg TE/g DE by 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and a high content of polyphenols, namely, hydroxycinnamic acids (neochlorogenic and p-coumaric acids) and anthocyanins (cyanidin-3-rutinoside and cyanidin-3-glucoside), compared with those of conventional extractions with low and high temperature and ultrasound-assisted extraction. The antioxidant extract produced from MAE could be a new alternative for the valorisation of non-compliant cherries since these extracts proved to be a functional ingredient due to the high content of antioxidants, which are linked to the prevention of diseases.
- Optimization of bromelain isolation from pineapple byproducts by polysaccharide complex formationPublication . Campos, Débora A.; Coscueta, Ezequiel R.; Valetti, Nadia Woitovich; Pastrana-Castro, Lorenzo M.; Teixeira, José A.; Picó, Guillermo A.; Pintado, Maria ManuelaA simple method for bromelain extraction from industrial pineapple residues (stems and peels) was developed and optimized through factorial experimental design. The developed methodology, based on precipitation with carrageenan, represents an alternative to the use of organic solvents and inorganic salts (common industrial precipitation) and allows achieving extracts with high bromelain purity. High recovery yield – 80–90% - of active bromelain was obtained for both crude juices (stems and peels) making possible to obtain ca. 0.3 g of bromelain from 100 g of pineapple byproducts using a low polysaccharide concentration (0.2–0.3% w/v).
- Study of olive pomace antioxidant dietary fibre powder throughout gastrointestinal tract as multisource of phenolics, fatty acids and dietary fibrePublication . Ribeiro, Tânia; Campos, Débora; Oliveira, Ana; Nunes, João; Vicente, António A.; Pintado, ManuelaPulp-enriched powder (POPP) was obtained from olive pomace solid fraction, a derived from the new value chain established for olive by-products. As a multifunctional powder, POPP retains several bioactive compounds (fatty acids, dietary fibre and phenolics) under potential synergic interaction, even more, reactive throughout the digestion. So, in this study, the potential multifunctionality of POPP was evaluated after the gastrointestinal tract. A significant loss of phenolics occurred during oral digestion (62.48 %). However, the potential role of dietary fibre as phenolics’ carrier and its possible liberation in the stomach allowed recovering a significant amount of phenolics (77.11 %) and a bioaccessibility index of at least 50 % (mainly for tyrosol and its glucoside). POPP also provides high content of dietary fibre mainly insoluble fibre (69.68 g/100 g dry weight) linked to a substantial amount of bound phenolics (7.63 mg of gallic acid equivalents/g fibre dry weight), with a positive effect on the fatty acids bioaccessibility [decreased the saturated (5-6 %) and facilitated the unsaturated fatty acids bioaccessibility (4-11%)]. PCA analysis became evident the negative effect of simulated gastrointestinal digestion upon POPP as mainly linked to phenolics’ loss. Despite all negative effects of the simulated digestion on POPP bioactive composition, phenolics and unsaturated fatty acids showed to be bioaccessible in significant amount, and the amount of bound phenolics associated to fibre retained in the colon have the potential to exert gut health benefits.