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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.
- Features and performance of edible films, obtained from whey protein isolate formulated with antimicrobial compoundsPublication . Ramos, Óscar L.; Silva, Sara I.; Soares, José C.; Fernandes, João C.; Poças, M. Fátima; Pintado, Manuela E.; Malcata, F. XavierThe goal of this research effort was to assess the efficacy of edible films produced fromwhey protein isolate (WPI) and glycerol, including incorporation of lactic acid (LA) and propionic acid (PRO), chitooligosaccharideswith nominal MW of 3 kDa (COS) and natamycin (NA) as antimicrobial agents. Their features were evaluated in vitro via agar diffusion and viable cell counting, against spoilage microflora often found contaminating cheese surfaces. The effect of incorporating the aforementioned compounds upon thickness, moisture content (MC), solubility (S), density (ρs),water activity (aw) and water vapor permeability (WVP), as well as upon tensile and optical properties of those films were also evaluated. Films formulated with LA, PRO or COS exhibited antimicrobial activity against all microorganisms tested, yet the viable cell count assay was more sensitive and reproducible. COS was the most active against Gram-negative bacteria, whereas LA was the most active against Gram-positive ones. NA was not active against bacteria, but displayed the strongest effect against yeasts. Incorporation of said antimicrobial compounds did not significantly (pN0.05) affect film thickness, yet it significantly (pb0.05) reduced tensile strength (TS). Incorporation of LA and NA in particular did not significantly (pb0.05) affect MC, S, ρs, WVP, elongation at break (EB) and Young's modulus (YM) values; however, a statistically significant increase (pb0.05) of MC, S andWVP, together with a statistically significant decrease (pb0.05) of ρs were attained upon incorporation of PRO or COS. Moreover, PRO produced the highest variation (pb0.05) in EB, TS and YM,whereas COS produced the highest change (pb0.05) in optical properties.
- Effect of composition of commercial whey protein preparations upon gelation at various pH valuesPublication . Ramos, Óscar S.; Pereira, Joana O.; Silva, Sara I.; Amorim, Maria M.; Fernandes, João C.; Lopes-da-Silva, José A.; Pintado, Manuela E.; Malcata, F. XavierThe major goal of this research effort was to comprehensively characterize various whey protein products available in the market — including one whey protein isolate (WPI) and three whey protein concentrates (two forms of WPC 80, and WPC 50), with regard to the effects of specific components (e.g. lecithin and minerals) and concentration of β-lactoglobulin (β-Lg) and α-lactalbumin upon thermal and gelation properties at various pH values (using micro differential scanning calorimetry, μDSC, and oscillatory rheometry). At pH values far from the isoelectric point of whey proteins, denaturation and aggregation appeared as one single endothermic peak in the corresponding μDSC heating thermograms, for WPI and both WPC 80; however, they appeared as separate transitions at pH 5. Acidic conditions increased the temperature of occurrence of the dominant endothermic transition associated to β-Lg, thus increasing the thermal stability of WPI, WPC 80A and WPC 80B. Gelation took place at the lowest temperature when pH was set at 5. WPI, WPC 80A and WPC 80B exhibited the highest G′ values at pH 5 — whereas WPI led to stronger gels than WPC, irrespective of pH. In the case of WPC 50, gelation did not occur at all.
- Natural extracts into chitosan nanocarriers for rosmarinic acid drug deliveryPublication . Silva, Sara Baptista da; Amorim, Manuela; Fonte, Pedro; Madureira, Raquel; Ferreira, Domingos; Pintado, Manuela; Sarmento, BrunoContext: Nanotechnology can be applied to deliver and protect antioxidants in order to control the oxidative stress phenomena in several chronic pathologies. Chitosan (CS) nanoparticles are biodegradable carriers that may protect antioxidants with potent biological activity such as rosmarinic acid (RA) in Salvia officinalis (sage) and Satureja montana (savory) extracts for safe and innovative therapies. Objective: Development and characterization of CS nanoparticles as a stable and protective vehicle to deliver RA for medical applications using natural extracts as sage and savory. Materials and methods: Antioxidant-CS based nanoparticles were prepared by ionic gelation with sodium tripolyphosphate (TPP), at pH 5.8 with a mass ratio of 7:1 (CS:TPP), with a theoretical antioxidant-CS loading of 40-50%. The nanoparticles were then characterized by different methods such as photon correlation spectroscopy, laser Doppler anemometry, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR), high-performance liquid chromatographic (HPLC), association efficiency, and antioxidant activity. Results and discussion: Individual and small sizing nanoparticles, around 300 nm, were obtained. SEM confirmed smooth and spherical nanoparticles after freeze-drying. No chemical interactions were found between antioxidants and CS, after encapsulation, by DSC and FTIR. The association efficiency was 51.2% for RA (with 40% loading) and 96.1 and 98.2% for sage and savory nanoparticles, respectively (both with 50% loading). Antioxidant activity values were higher than 0.0348 eq [Asc. Ac.] g/L/g extract and 0.4251 μmol/eq Trolox/g extract. Conclusion: The extracts under study are promising vehicles for RA drug delivery in CS nanocarriers.
- Chitosan-based nanoparticles for rosmarinic acid ocular delivery-in vitro testsPublication . Silva, Sara; Ferreira, Domingos; Pintado, M. E.; Sarmento, BrunotIn this study, chitosan nanoparticles were used to encapsulate antioxidant rosmarinic acid, Salvia offici-nalis (sage) and Satureja montana (savory) extracts as rosmarinic acid natural vehicles. The nanoparticleswere prepared by ionic gelation using chitosan and sodium tripolyphosphate (TPP) in a mass ratio of7:1, at pH 5.8. Particle size distribution analysis and transmission electron microscopy (TEM) confirmedthe size ranging from 200 to 300 nm, while surface charge of nanoparticles ranged from 20 to 30 mV.Nanoparticles demonstrate to be safe without relevant cytotoxicity against retina pigment epithelium(ARPE-19) and human cornea cell line (HCE-T). The permeability study in HCE monolayer cell line showedan apparent permeability coefficient Pappof 3.41 ± 0.99 × 10−5and 3.24 ± 0.79 × 10−5cm/s for rosmarinicacid loaded chitosan nanoparticles and free in solution, respectively. In ARPE-19 monolayer cell line thePappwas 3.39 ± 0.18 × 10−5and 3.60 ± 0.05 × 10−5cm/s for rosmarinic acid loaded chitosan nanoparti-cles and free in solution, respectively. Considering the mucin interaction method, nanoparticles indicatemucoadhesive proprieties suggesting an increased retention time over the ocular mucosa after instilla-tion. These nanoparticles may be promising drug delivery systems for ocular application in oxidative eyeconditions.
- Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefromPublication . Ramos, Óscar L.; Reinas, Isabel; Silva, Sara I.; Fernandes, João C.; Cerqueira, Miguel A.; Pereira, Ricardo N.; Vicente, António A.; Poças, M. Fátima; Pintado, Manuela E.; Malcata, F. XavierThis manuscript describes the detailed characterization of edible films made from two different protein products e whey protein isolate (WPI) and whey protein concentrate (WPC), added with three levels of glycerol (Gly) e i.e. 40, 50 and 60%(w/w). The molecular structure, as well as barrier, tensile, thermal, surface and optical properties of said films were determined, in attempts to provide a better understanding of the effects of proteinaceous purity and Gly content of the feedstock. WPI films exhibited statistically lower (p < 0.05) moisture content (MC), film solubility (S), water activity, water vapor permeability (WVP), oxygen and carbon dioxide permeabilities (O2P and CO2P, respectively) and color change values, as well as statistically higher (p < 0.05) density, surface hydrophobicity, mechanical resistance, elasticity, extensibility and transparency values than their WPC counterparts, for the same content of Gly. These results are consistent with data from thermal and FTIR analyses. Furthermore, a significant increase (p < 0.05) was observed in MC, S, WVP, O2P, CO2P, weight loss and extensibility of both protein films when the Gly content increased; whereas a significant decrease (p < 0.05) was observed in thermal features, as well as in mechanical resistance and elasticity e thus leading to weaker films. Therefore, fundamental elucidation was provided on the features of WPI and WPC germane to food packaging e along with suggestions to improve the most critical ones, i.e. extensibility and WVP.
- Evaluation of antimicrobial edible coatings from a whey protein isolate base to improve the shelf life of cheesePublication . Ramos, Ó. L.; Pereira, J. O.; Silva, S. I.; Fernandes, J. C.; Franco, M. I.; Lopes-da-Silva, J. A.; Pintado, M. E.; Malcata, F. X.The objective of this work was to evaluate the effectiveness of antimicrobial edible coatings to wrap cheeses, throughout 60 d of storage, as an alternative to commercial nonedible coatings. Coatings were prepared using whey protein isolate, glycerol, guar gum, sunflower oil, and Tween 20 as a base matrix, together with several combinations of antimicrobial compounds—natamycin and lactic acid, natamycin and chitooligosaccharides (COS), and natamycin, lactic acid, and COS. Application of coating on cheese decreased water loss (~10%, wt/wt), hardness, and color change; however, salt and fat contents were not significantly affected. Moreover, the antimicrobial edible coatings did not permit growth of pathogenic or contaminant microorganisms, while allowing regular growth of lactic acid bacteria throughout storage. Commercial nonedible coatings inhibited only yeasts and molds. The antimicrobial edible coating containing natamycin and lactic acid was the best in sensory terms. Because these antimicrobial coatings are manufactured from food-grade materials, they can be consumed as an integral part of cheese, which represents a competitive advantage over nonedible coatings.
- In situ crosslinked electrospun gelatin nanofibers for skin regenerationPublication . Dias, J. R.; Baptista-Silva, S.; Oliveira, C. M. T. de; Sousa, A.; Oliveira, Ana L.; Bártolo, P. J.; Granja, P. L.Due to its intrinsic similarity to the extracellular matrix, gelatin electrospun nanofibrous meshes are promising scaffold structures for wound dressings and tissue engineering applications. However, gelatin is water soluble and presents poor mechanical properties, which generally constitute relevant limitations to its applicability. In this work, gelatin was in situ crosslinked with 1,4-butanediol diglycidyl ether (BDDGE) at different concentrations (2, 4 and 6 wt%) and incubation time-points (24, 48 and 72 h) at 37 °C. The physico-chemical and biological properties of BDDGE-crosslinked electrospun gelatin meshes were investigated. Results show that by changing the BDDGE concentration it is possible to produce nanofibers crosslinked in situ with well-defined morphology and modulate fiber size and mechanical properties. Crosslinked gelatin meshes show no toxicity towards fibroblasts, stimulating their adhesion, proliferation and synthesis of new extracellular matrix, thereby indicating the potential of this strategy for skin tissue engineering.
- Edible films and coatings from whey proteins: a review on formulation, and on mechanical and bioactive propertiesPublication . Ramos, Óscar L.; Fernandes, João C.; Silva, Sara I.; Pintado, Manuela E.; Malcata, F. XavierThe latest decade has witnessed joint efforts by the packaging and the food industries to reduce the amount of residues and wastes associated with food consumption. The recent increase in environmental awareness has also contributed toward development of edible packaging materials. Viable edible films and coatings have been successfully produced from whey proteins; their ability to serve other functions, viz. carrier of antimicrobials, antioxidants, or other nutraceuticals, without significantly compromising the desirable primary barrier and mechanical properties as packaging films, will add value for eventual commercial applications. These points are tackled in this review, in a critical manner. The supply of whey protein-based films and coatings, formulated to specifically address end-user needs, is also considered.
- Biomechanical performance of hybrid electrospun structures for skin regenerationPublication . Dias, J. R.; Baptista-Silva, S.; Sousa, A.; Oliveira, A. L.; Bártolo, P. J.; Granja, P. L.Wound dressings made by electrospun nanofibers have been demonstrating great potential to regenerate skin tissue as compared to the conventional membrane products available in the market. Until today most of the developed dressings have only demonstrated the capability to regenerate the dermis or epidermis. In this study we propose new hybrid electrospun meshes combining polycaprolactone and gelatin. Several approaches, multilayer, coating and blend were stablished to investigate the most appropriate hybrid structure with potential to promote skin regeneration in its full thickness. The structures were evaluated in terms of physico-chemical properties (porosity, water vapor permeability, contact angle and swelling degree) and according to its mechanical and biological performance. Multilayer and blend structures demonstrated to fit most of native skin requirements. However, looking to all the performed characterization we considered multilayer as the most promising hybrid structures, due its high porosity which contributed to an ideal water vapor permeability rate and good mechanical and biological properties. Based on this multilayer structure is a promisor wound dressing.