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Production of a food grade blueberry extract rich in anthocyanins: selection of solvents, extraction conditions and purification method

Publication . Silva, Sara; Costa, Eduardo M.; Calhau, Conceição; Morais, Rui M.; Pintado, M. Manuela E.

Blueberries are recognized, by the scientific community and consumers, for their health promoting potential. This fact makes blueberries, and blueberry derived products, prime candidates to aid in the development of healthier foodstuffs that are easily recognized as such by consumers. As blueberries health promoting properties are frequently associated with their phenolic, particularly anthocyanins, composition the present work aimed to establish a simple, food safe, approach to extract these compounds. One that, while being food safe also allowed for a relatively easy scale up process. To accomplish this, four different solvents (water, ethanol, methanol and acetone) acidified or not, were evaluated for their extraction capabilities. The results obtained demonstrated that ethanol acidified with 0.01% HCl was an effective extractant of both phenolic compounds and anthocyanins. Acetone was significantly more effective in extracting phenolic compounds but it was almost unable to extract anthocyanins, with the addition of acid allowing for an increase of anthocyanin yield but reducing the overall phenolic yield. The extraction of anthocyanins may be further improved with the addition of an ultrasound treatment to the extraction process, though no significant differences were observed when considering the overall phenolic content. Furthermore, if seeking a condensed extract, purification using solid phase extraction columns allowed the production of an extract comprised of ca. 40% (w w(-1)) anthocyanins.

2017Journal article

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DNA agarose gel electrophoresis for antioxidant analysis: Development of a quantitative approach for phenolic extracts

Publication . Silva, Sara; Costa, Eduardo M.; Vicente, Sandra; Veiga, Mariana; Calhau, Conceição; Morais, Rui M.; Pintado, Manuela E.

Most of the fast in vitro assays proposed to determine the antioxidant capacity of a compound/extract lack either biological context or employ complex protocols. Therefore, the present work proposes the improvement of an agarose gel DNA electrophoresis in order to allow for a quantitative estimation of the antioxidant capacity of pure phenolic compounds as well as of a phenolic rich extract, while also considering their possible pro-oxidant effects. The result obtained demonstrated that the proposed method allowed for the evaluation of the protection of DNA oxidation [in the presence of hydrogen peroxide (H2O2) and an H2O2/iron (III) chloride (FeCl3) systems] as well as for the observation of pro-oxidant activities, with the measurements registering interclass correlation coefficients above 0.9. Moreover, this method allowed for the characterization of the antioxidant capacity of a blueberry extract while demonstrating that it had no perceived pro-oxidant effect.

2017Journal article

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Quercus based coffee-like beverage: effect of roasting process and functional characterization

Publication . Coelho, Marta; Silva, Sara; Rodríguez-Alcalá, Luis Miguel; Oliveira, Ana M.; Costa, Eduardo; Borges, André; Martins, Célia; Rodrigues, António S.; Pintado, Maria Manuela E.

Coffee is one of the world’s most widely consumed beverages but intake it is not encouraged in consumers with some health conditions. In this way, with the growing interest in developing healthier substitutes, a coffee-like beverage obtained from Quercus ilex and Quercus suber acorn´s species, was elaborates maintaining the flavor. Although, this beverage is a promising coffee alternative, little is known about effects of roasting process in its composition. To that end the antioxidant capacity and toxicity of the developed coffee were analyzed and phenolic compounds and fatty acids (esterified and free forms) were characterized through HPLC-DAD and GC-FID, respectively. The results showed that Quercus based beverages presented antioxidant capacity related to their phenolic content, mainly to ellagic acid as the primary phenolic compound identified. Due to this composition, the beverage also presented antimutagenic activity. The main fatty acids in the esterified lipids were mainly oleic, linoleic, palmitic, stearic and cis vaccenic. In the free fatty acids fraction (FFA) they were oleic, linoleic and palmitic acids. Heat processing produced a reduction in total fatty acid concentration in TG and FFA fraction of Q. ilex. Nevertheless, for Q. suber alterations were only found for FFA. Coffee production did not form genotoxic or cytotoxic compounds. Overall, these results show the feasibility of Quercus acorn-based foodstuffs and its potential to produce a functional coffee-like beverage.

2018Journal article

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Insights into chitosan antibioﬁlm activity against methicillin-resistant Staphylococcus aureus

Publication . Costa, E. M.; Silva, S.; Tavaria, F. K.; Pintado, M. M.

Aims: Chitosan is a natural compound that has been validated as a viable antimicrobial agent against Staphylococcus aureus. With this work we sought to evaluate the planktonic and sessile sensitivity of methicillin-resistant S. aureus to chitosan's activity and evaluate if methicillin-resistant S. aureus (MRSA) would be more or less sensitive to chitosan's activity than methicillin-sensitive S. aureus (MSSA). Methods and Results: A group comprised of reference strains and clinical multiresistant isolates of MSSA and MRSA were used. Methicilin resistance effect upon chitosan activity was assessed in planktonic setting and in different phases of sessile colonization, namely adhesion, biofilm formation and mature biofilm through biomass and metabolism inhibition. The results obtained showed that S. aureus methicillin resistance mechanism did not impair chitosan's activity as the highest bacterial susceptibility was registered for MRSA. Chitosan was highly effective in inhibiting MSSA and MRSA strains in both planktonic and sessile settings with biofilm inhibition percentages reaching as high as 90% for MRSA. Conclusions: Staphylococcus aureus methicillin resistance did not impair chitosan's antimicrobial and antibiofilm activities and MRSA and MSSA were inhibited both in planktonic and sessile settings at low concentrations with great efficacy. Significance and Impact of the Study: Considering the obtained results chitosan shows potential as an alternative for the control of biofilm-related recalcitrant MRSA infections.

2017Journal article

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Textile dyes loaded chitosan nanoparticles: characterization, biocompatibility and staining capacity

Publication . Costa, Eduardo M.; Silva, Sara; Veiga, Mariana; Baptista, Patricia; Tavaria, Freni K.; Pintado, Manuela E.

Textile dyeing is a hazardous and toxic process. While traditionally it has been managed through effluent treatment, new approaches focused upon improving the dyeing process are gaining relevance. In this work, we sought to obtain, for the first time, an eco-friendly chitosan-nanoparticle based textile dyeing method. To that end, yellow everzol and navy blue itosperse loaded chitosan nanoparticles were produced and their capacity to dye textiles and cytotoxicity towards human skin cells were evaluated. The results obtained showed that it was possible to obtain nanoencapsulated dyes through ionic gelation with an average entrapment efficacy above 90 %. Nanoparticles presented a positive surface charge and sizes between 190 and 800 nm with yellow everzol NPs occurring via ionic interactions while navy blue itosperse NPs were formed through hydrogen bonds. Furthermore, the produced dye NPs presented no cytotoxicity towards HaCat cells and presented staining percentages reaching 17.60 % for a viscose/wool blend.

2021Journal article

Open access