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- Development and validation method for simultaneous quantification of phenolic compounds in natural extracts and nanosystemsPublication . Silva, Sara Baptista da; Oliveira, Ana; Ferreira, Domingos; Sarmento, Bruno; Pintado, ManuelaIntroductionSage and savoury (Salvia sp. and Satureja montana, respectively) are plants used in traditional medicine. The quality control of their herbal formulations is of paramount concern to guarantee the expected biological activity of their anti-oxidant compounds. Objective To establish a simple and effective high-performance liquid chromatographic (HPLC) method to evaluate simultaneously quercetin and rosmarinic acid, in a pure form, in natural extracts (sage and savoury), and encapsulated into chitosan nanoparticles. Methods Chromatography was performed on an RP C-18-column, in a gradient mode with a mobile phase comprising methanol:formic acid:water 92.5:2.5:5 (v/v) at a flow rate of 0.75mL/min and at wavelength of 280nm. Results The method was specific, linear in the range of 0.05-1mg/mL (R-2=1.00), precise at the intraday and interday levels, accurate (recovery rate 90.50.6%), and robust to changes in equipment conditions. Conclusion The method established was effective for quercetin and rosmarinic acid characterisation in natural extracts and in chitosan nanoparticles, allowing the loading capacity determination, the association efficiency as well as the in vitro release. This study shows a new HPLC method validated to simultaneous quantification of rosmarinic acid and quercetin in natural extracts and chitosan nanoparticles. This method is also able for detection and quantification of other phenolic and flavanoid compounds in natural matrix.
- Effect of modified atmosphere on phytochemical profile of pasteurized peach puréesPublication . Oliveira, Ana; Coelho, Marta; Alexandre, Elisabete; Gomes, Maria Helena; Almeida, Domingos P. F.; Pintado, ManuelaPeach phytochemical profile was analysed during 90 days storage under three different gas mixtures: 10 kPa O2+90 kPa N2, 100 kPa N2 and air (78 kPa N2+21 kPa O2+0.03 kPa CO2) for both 4 and 23 °C. The antioxidant activity increased when peach purée was stored under air at 4 °C and 100 kPa N2 at 23 °C, while total phenolic content was not affected by atmospheres. Total carotenoids were more stable for 100 kPa N2, where it decreases 48 and 58% respectively for 4 and 23 °C. The content of (+)-catechin decreased 42% for 10 kPa O2 at 4°C, remaining constant in other atmospheres. Chlorogenic and neochlorogenic acids were well preserved in atmospheres containing oxygen, presenting an increase of 14 and 24% for 10 kPa O2 at 4°C. The higher content of carotenoids was obtained for 100 kPa N2 at 23 °C. Principal component analysis shows that atmospheres variance was associated with carotenoids while storage time was more related with total phenolics and total antioxidant capacity.
- The effect of sex, season and gametogenic cycle on gonad yield, biochemical composition and quality traits of Paracentrotus lividus along the North Atlantic coast of PortugalPublication . Rocha, Filipa; Baião, Luís F.; Moutinho, Sara; Reis, Bruno; Oliveira, Ana; Arenas, Francisco; Maia, Margarida R. G.; Fonseca, Antonio J. M.; Pintado, Manuela; Valente, Luisa M. P.Sea urchin population harvest in the North Atlantic coast of Portugal was characterized in terms of gonad yield, nutritional composition and important market-related traits, over one reproductive cycle (March 2016 to March 2017). Most of the quality attributes showed a seasonal variation strongly dependent on sea urchin sex. Maximum gonad yield (18%) was observed in March 2017. A single spawning event occurred between May and July. Gonads are rich sources of protein (12–18% WW) with low fat content (≤6% WW), that increase during the gametogenic stages of recovery and growing (November-December). Polyunsaturated fatty acids were the dominant class in both sexes (4.2–14.7 mg.g−1 WW), being preferentially accumulated in females. Total gonads carotenoid varied seasonally, with the highest level being observed in males during spawning season. Echinenone was the main pigment present in gonads, showing highest concentrations in males during spawning and gonad recovering. During the growing and early maturation period gonads were more reddish, yellowish and brighter, as well as more firm, irrespectively of the sex. Based on all seasonal changes affecting gonad yield and quality, the period between November and February seems the most suitable to harvest high quality gonads in the Atlantic coast of Portugal.
- 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%.
- Long-term frozen storage and pasteurization effects on strawberry polyphenols contentPublication . Oliveira, Ana; Coelho, Marta; Alexandre, Elisabete M. C.; Almeida, Domingos P. F.; Pintado, ManuelaThe profile of the strawberry polyphenols was analysed on fresh strawberry, immediately after freezing or after pasteurization and during frozen storage of strawberry at -20 A degrees C for 360 days. Freezing increased the extractability of (-)-epigallocatechin gallate (60 %), (-)-epicatechin (48 %) and ellagic acid (51 %) and decreased the pelargonidin-3-glucoside (18 %). Direct pasteurization of fresh strawberry increased the extractability for (-)-epigallocatechin gallate (73 %), (-)-epicatechin (45 %) and ellagic acid (143 %) and decreased for pelargonidin-3-glucoside (16 %). During storage at -20 A degrees C for 360 days the extractable levels of pelargonidin-3-rutinoside, (-)-epigallocatechin gallate, (+)-catechin, (-)-epicatechin and ellagic acid decreased by 17, 20, 37, 65 and 80 %, respectively, while those of pelargonidin-3-glucoside increased 13 %. Changes in polyphenols content, produced by pasteurization after 360 days frozen storage, were minimal. Polyphenols content decreased during storage of strawberry pieces at -20 A degrees C for 360 days, while the thermal treatment applied after freezing storage did not induce any significant change.
- Non-compliant fruit as new functional food ingredientsPublication . Vilas-Boas, Ana A.; Gómez-García, Ricardo; Campos, Débora A.; Oliveira, Ana; Pintado, ManuelaOver the last decade, the high amount of food waste has received increased attention towards a more sustainable future, such as reducing food losses and valorize food by-products. Non-compliant fruit represents a significant part of fruit losses during production as a result of aesthetic requirements (color, low size, and deformations in the shape of the products). These fruits do not match commercialization standards when sold as fresh fruit. Selling this kind of fruit at a cheap price might have a great impact on the reduction of fruit loss, nevertheless, this option does not offer a long-time solution, being required an alternative and sustainable approach. These fruits represent a rich source of bioactive compounds (phenolic compounds and dietary fiber) that could be used as new functional ingredients/products. The production of new functional ingredients/products from non-compliant fruit consists of a valuable and sustainable approach for taking advantage of these food losses. Therefore, the purpose of this chapter is to provide an overview of new strategies to recover from non-compliant fruit value-added ingredients, impacting society, environment, and economy.
- Impact of processing on nutritional and functional properties of strawberryPublication . Oliveira, Ana; Pintado, M. E.The market of processed fruits has grown rapidly in recent decades as a result of changes in consumer preferences and attitudes. Strawberry is one of most preferred fruits to incorporate in milk based products or other derived-products. Strawberry antioxidant capacity is affected by the different post-harvest features, such as processing and storage conditions (e.g., time, temperature). Although reducing quality, these processing conditions are required for safety, quality and economic reasons. In order to assure microbial stability some thermal processing techniques can be applied. These techniques as well storage conditions after processing can affect the phytochemicals composition and nutritional quality of fruit by promoting changes resulting from cell wall and membrane disruption and chemical reactions. Thermal processing of fruits involves heating to temperatures ranging from 50 to 150 ºC, depending on pH of the product and desired shelf-life. Such treatments may cause undesirable biochemical changes that affect the sensory and nutritional quality of the final product. Antioxidants play a key role in the prevention of oxidative stress-related diseases, and the reduction of chronic diseases has been associated to diets rich in plant foods, particularly fruits. Strawberries are a good source of ascorbic acid, anthocyanins and flavonols, and among the fruits, they possess one of the highest antioxidant activities. Among the phytochemicals, phenols, have received a great deal of attention because of their antioxidant activity, which help to neutralise free radicals. Strawberries are a good source of polyphenols such as tannins, ellagic acid and anthocyanins. Thermal treatment is one of the most important factors affecting the stability of phenolic compounds. During processing and subsequent storage, phenolic content and antioxidant activity is changed mainly associated to thermal degradation of anthocyanins, but also due to chemical reactions occurring during storage of processed fruit promoted by temperature but also by other factors such as oxygen present in air, acidity of the medium, presence of enzymes or metals. The aim of this chapter, therefore, is to review the impact of different thermal techniques and associated storage conditions (e.g., time and temperature) of processed strawberry on nutritional and functional characteristics of strawberries, and establish their role on the quality of fruit.
- Optimization of two biopolymer-based oral films for the delivery of bioactive moleculesPublication . Castro, Pedro M.; Fonte, Pedro; Oliveira, Ana; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Manuela E.An experimental design was established in order to optimize the mechanical properties of two oral film formulations intended for oral delivery of bioactive compounds. Carboxymethylcellulose (CMC) and gelatin type A (GelTA) were selected as polymeric matrix. Scanning electron microscopy revealed that caffeine crystals were homogeneously dispersed onto oral film matrix. Fourier-transform infrared analysis did not indicate formation of new chemical entities. USP modified dissolution assay revealed that GelTA was more effective in controlling caffeine release since maximum caffeine release (97.4%±0.95) after 20min. On the other hand, CMC is better indicated for immediate release since maximum caffeine release (81.1%±2.14) occurred after 4min. Simulation of gastrointestinal tract with ex vivo permeability assay was in accordance with USP dissolution assay (42.0%±7.79 and 15.3%±4.0 of caffeine released from CMC and GelTA oral films (OF), respectively, permeated porcine intestinal mucosa after 120min). CMCOF and GelTAOF optimized formulations represent two suitable oral delivery systems for immediate and controlled release, respectively.
- Combinatory approach for developing silk fibroin-based scaffolds with hierarchical porosity and enhanced performance for cartilage tissue engineering applicationsPublication . Ribeiro, Viviana P.; Yan, Le-Ping; Oliveira, Ana; Oliveira, Joaquim M.; Reis, Rui L.Introduction: The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication and for TE applications, including for cartilage defects repair[1]. Recently, it was found that proteins like silk fibroin (SF), presenting tyrosine groups can be used to prepare fast formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)[2],[3]. Moreover, the high versatility, processability and tailored mechanical properties of SF, make this natural polymer attractive for the development of innovative scaffolding strategies for cartilage TE applications[4],[5]. Materials and Methods: The present work proposes a novel route for developing SF-based scaffolds derived from highconcentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex. The combination of salt-leaching and freeze-drying methodologies was used to prepare macro/microporous SF scaffolds with an interconnected structure and specific features regarding biodegradation and mechanical properties (Fig. 1a). The scaffolds morphology and porosity were analyzed by SEM and micro-CT. The mechanical properties (Instron) and protein conformation (FTIR, XRD) were also assessed. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 day. This work also aims to evaluate the in vitro chondrogenic differentiation response by culturing human adipose derived stem cells (hASCs) over 21 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro/microporous structures will be evaluated through different quantitative (Live/Dead, DNA, GAGs, RT PCR) and qualitative (SEM, histology, immunocytochemistry) assays. Results and Discussion: The macro/microporous SF scaffolds showed high porosity and interconnectivity with the trabecular structures evenly distributed (Fig. 1b,c). A dramatic decrease of compressive modulus was observed for samples in hydrated state. Chemical analysis revealed that SF scaffolds displayed the characteristic peaks for β-sheet conformation. Swelling ratio data demonstrated a large swelling capacity, maintaining their structural integrity for 30 days. As expected, when immersed in protease XIV the degradation rate of SF scaffolds increased. Based on the promising morphology and physicochemical properties of the developed SF scaffolds, in vitro chondrogenic differentiation studies with hASCs are envisioned in order to validate their performance for cartilage regeneration applications.Conclusion: This study proposes an innovative approach to produce fast-formed porous SF scaffolds using enzymatically crosslinked SF hydrogels structured by the combination of salt-leaching and freeze-drying methodologies. The obtained results can provide a valuable reference of SF as a tunable and versatile biomaterial with great potential for applications in cartilage TE scaffolding.
- Phytochemicals preservation in strawberry as affected by pH modulationPublication . Oliveira, Ana; Gomes, Maria Helena; Alexandre, Elisabete M. C.; Poças, Fátima; Almeida, Domingos P. F.; Pintado, ManuelaStrawberries purees are incorporated in foods and subjected to pH modulation according to the expected final food matrix. The effect of pH on strawberry polyphenols stored at 4 and 23 degrees C for 90 days was evaluated. Total antioxidant activity and total phenolics content were only affected by time according to a first order model. The pH 4.5 induced higher decrease in (-)-epigallocatechin gallate (71% and 79%) and quercetin-3-glucoside (29% and 36%), for both storage temperatures. For pH 2.5 and 3.0, ellagic acid increased 84% for 4 degrees C. and 185% for 23 degrees C. Anthocyanins concentration changes along storage were well described by first order model. The pH value of 2.5 presented the lower kinetic constant rate where cyanidin-3-glucoside, pelargonidin-3-glucoside and pelargonidin-3-rutinoside had a k = 0.04, 0.05 and 0.03 day(-1). Lower storage temperature (4 degrees C) and lower pH (2.5) were the best condition for the preservation of polyphenols in pasteurized strawberry during a 90-day storage period.