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The strawberry tree (Arbutus unedo) is an evergreen plant belonging to the Ericaceae family, an endemic specie of the Mediterranean. In Portugal it grows mostly wild and its fruits are mainly used to produce a distillated alcoholic beverage called “Aguardente de Medronho”. From the process, a residue is obtained which is normally discarded, despite it may figure as biomass for valorization. This work intends to study different conditions of supercritical fluid extraction (SFE), using carbon dioxide as solvent and ethanol as co-solvent for the recovery of bioactive compounds from that residue. After a screening design of experiments varying pressure, temperature, co-solvent, flow rate and time of extraction, an optimization design of experiments (central composite design) was performed. Time (90 min) and flow rate (10 g/min) were established. Variables were pressure (100, 200 and 300 bar), temperature (35, 45 and 55 °C) and co-solvent concentration (0, 10 and 20 %). Extracts were analyzed by GC–MS and LC–MS/MS and cytotoxicity and antiproliferative effect of the extracts was evaluated in Caco2 and HT-29 cell models respectively. Chemical characterization showed an increase in phenolic compounds such as pyrogallol, gallic acid, catechol and protocatechuic acid amongst others, with the increase of the co-solvent. GC–MS analysis revealed a diversity of compounds from fatty acid esters, terpenes and small molecules with recognized bioactivity. The most promising extract was obtained using 300 bar and 55 °C and 20 % of co-solvent. Besides being the extract with the highest yield, it also presented the lowest EC50 (0.569 mg/mL) mainly due to the presence of phenolic compounds.

Bravo de Esmolfe (BE) is a traditional Portuguese apple highly appreciated by consumers due to its peculiar flavor and aroma. This apple contains higher concentration of phenolic compounds than other cultivars and is thus considered a rich source of antioxidants. Its sensorial and functional properties have attracted farmers’ associations to increase BE production. However, a large quantity of apples is wasted due to storage/transportation procedures that impact BE’s quality attributes. In this work, we applied high-pressure extraction methodologies to generate antioxidant-rich fractions from BE residues aiming at adding high value to these agro-food by-products. We performed a first extraction step using supercritical CO2, followed by a second extraction step where different CO2 + ethanol mixtures (10–100% v/v) were tested. All experiments were carried out at 25 MPa and 50 °C. Extracts were characterized in terms of global yield, phenolic content and antioxidant activity using chemical (ORAC, HOSC, HORAC) and cell-based assays (CAA). We demonstrated that, although the pressurized 100% ethanol condition promoted the highest recovery of phenolic compounds (509 ± 8 mg GAE/100 g BE residues), the extract obtained with 40% ethanol presented the highest CAA (1.50 ± 0.24 µmol QE/g dw) and ORAC (285 ± 16 µmol TEAC/g dw), as well as HOSC and HORAC values, which correlated with its content of epicatechin and procyanidin B2. Noteworthy, this fraction inhibited free radical production in human neurospheroids derived from NT2 cells, a robust 3D cell model for neuroprotective testing.

Curcumin nanoemulsions stabilized by whey protein isolate were successfully developed using high-pressure homogenization. The effect of a chitosan layer deposition using the layer-by-layer technique on nanoemulsions' stability was evaluated during storage conditions, as well as during gastrointestinal tract passage. Lipids hydrolysis and curcumin bioaccessibility was assessed using a dynamic gastrointestinal model (simulating the stomach, duodenum, jejunum and ileum) and the cytotoxicity, cellular antioxidant activity and permeability analyses were carried out using Caco-2cells. Results showed that both nanosystems were stable during one month of storage and at stomach pH conditions, whereas creaming and phase separation occurred at intestine pH conditions. The addition of a chitosan layer increased curcumin bioaccessibility, whereas cellular antioxidant activity studies revealed that nanoemulsions and multilayer nanoemulsions exhibited 9 and 10 times higher antioxidant capacity at the cellular level, respectively, when compared to free curcumin. Permeability assays showed that the use of a chitosan layer significantly increased the apparent permeability coefficient of curcumin through Caco-2cells by 1.55-folds., The authors Hélder D. Silva, Ana C. Pinheiro and Miguel A. Cerqueira (SFRH/BD/81288/2011, SFRH/BPD/101181/2014 and SFRH/BPD/72753/2010, respectively) are the recipients of a fellowship from the Foundation for Science and Technology. This work was supported by Portuguese Foundation for Science and Technology under the scope of the Project PTDC/AGR-TEC/5215/2014, of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, PEst-OE/EQB/LA0004/2011 and iNOVA4Health - UID/Multi/04462/2013, a program financially supported by FCT/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. The authors also acknowledge the European Commission: BIOCAPS (316265, FP7/REGPOT-2012-2013.1). The support of EU Cost Action FA1001 is gratefully acknowledged. The authors also acknowledge Stepan (The Netherlands) for providing the Neobee 1053 oil, and Rui Fernandes from IBMC, University of Porto for assistance in taking the TEM pictures., info:eu-repo/semantics/publishedVersion

An emergent research area in software engineering and software reliability is the use of wearable biosensors to monitor the cognitive state of software developers during software development tasks. The goal is to gather physiologic manifestations that can be linked to error-prone scenarios related to programmers’ cognitive states. In this paper we investigate whether electroencephalography (EEG) can be applied to accurately identify programmers’ cognitive load associated with the comprehension of code with different complexity levels. Therefore, a controlled experiment involving 26 programmers was carried. We found that features related to Theta, Alpha, and Beta brain waves have the highest discriminative power, allowing the identification of code lines and demanding higher mental effort. The EEG results reveal evidence of mental effort saturation as code complexity increases. Conversely, the classic software complexity metrics do not accurately represent the mental effort involved in code comprehension. Finally, EEG is proposed as a reference, in particular, the combination of EEG with eye tracking information allows for an accurate identification of code lines that correspond to peaks of cognitive load, providing a reference to help in the future evaluation of the space and time accuracy of programmers’ cognitive state monitored using wearable devices compatible with software development activities.

Arbutus unedo fruits, commonly known as strawberry-tree fruits, can be used as raw material for the development of bioactive ingredients, besides other traditional applications. In this work, a high-pressure CO2 Assisted Extraction process (HPCDAE) was investigated in order to find the best process conditions to obtain extracts with a higher phenolic content (TPC) and antioxidant activity. The process parameters studied were pressure (100, 175 and 250 bar), temperature (40 °C, 55 °C and 70 °C) and volume solid:liquid (S:L)/CO2 ratios (20, 50 and 80%) and the extracts recovered compared with the extract obtained with a S:L conventional extraction. Results show that when the highest pressure (250 bar) temperature (70 °C) and lowest S:L/CO2 ratio (20%) are used, HPCDAE increases 1.41 times TPC of the extracts, compared to conventional S-L extract. A significant increase in the extraction of galloyl hexoside and 5-O-galloylquinic acid were observed after HPCDAE application. The intensification of both compounds extraction may be related with the increase in the antioxidant activity of the extracts. The results from this study show that HPCDAE is a promising tool to intensify the extraction of bioactive compounds from strawberry tree fruits.

Background: Aiming at the enhancement of food products' nutritional and health value, the incorporation of nutraceuticals has attracted increasing interest in the last years. However, they often exhibit low water solubility and stability, limiting their direct incorporation into food products. Also, they show very low bioavailability due to limited bioaccessibility, poor absorption and/or chemical transformation within the gastrointestinal tract. This renders their health benefits extremely difficult to be realized by the consumers. Scope and Approach In the present review the recent innovations regarding the formulation and design of bio-based micro and nano-delivery systems to encapsulate nutraceuticals is discussed; it also gives an overview of the challenges associated to their development; and highlights some strategies to enhance nutraceuticals' bioavailability. An insight about delivery systems' potential toxicity (in particular at nano-scale) is also provided. Key Findings and Conclusions Recent developments in the design of bio-based delivery systems offer the possibility of stabilizing and enhancing nutraceuticals' functionality within food products. In fact, different strategies can be used to enhance nutraceuticals' bioavailability: i) nano-delivery systems, besides showing a huge potential for the protection of valuable nutraceuticals during food processing/digestion, can be used to increase their bioavailability; ii) absorption enhancement technologies have been successfully used to increase nutraceuticals' membrane permeation; and iii) excipient foods have been shown to improve nutraceuticals' biological activity. However, the application of these enabling technologies to food is hindered by very pertinent issues that can be summarized in the effective preservation/maximization of the nutraceuticals' bioactivity and safety, once inside the human body., Ana C. Pinheiro and Joana T. Martins acknowledge the Foundation for Science and Technology (FCT) for their fellowships (SFRH/BPD/ 101181/2014 and SFRH/BPD/89992/2012). This work was supported by Portuguese Foundation for Science and Technology (FCT) under the scope of the Project PTDC/AGR-TEC/5215/2014, of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-010145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

This study aimed to evaluate the antioxidant and antimicrobial activities of Melissa officinalis L. extracts. In order to estimate the best conditions to extract polyphenols from the plant studied, different solvents such as hexane, acetone, and ethanol were used. The polyphenols content was estimated by the Folin–Ciocalteu method. The antioxidant activity was assessed by different methods such as ORAC test and the scavenging of free radical DPPH, while ascorbic acid was used as a control. The antimicrobial activity of M. officinalis L. was assessed by the method of disk diffusion. The results obtained showed that ethanol was the most efficient solvent for extracting polyphenols from the M. officinalis L. The ethanol extract exhibited strong antioxidant and antimicrobial activities with an EC 50 of $$18.16 \pm 0.64\,\upmu \hbox {g}/ \hbox {mL}$$ and a strong inhibition against Staphylococcus aureus. The results obtained suggest that Algerian M. officinalis L. can be used as an important source of natural antioxidant and antimicrobial agent for food and pharmaceutical industries.

Quercetin is an antioxidant compound with highly promising pharmacological properties against a wide variety of diseases. A major limitation for the clinical application of quercetin is its low bioavailability, due to its low solubility in water. In this work, quercetin is microencapsulated in a surfactant material with the objective of increasing its bioavailability. For this, quercetin is first treated using Supercritical Fluid Extraction of Emulsions in order to precipitate it in nanometric scale in an aqueous suspension, and then this suspension is further treated by Particles from Gas Saturated Solutions (PGSS)-drying technology, to obtain quercetin loaded dried particles in micrometric scale. Soy-bean lecithin and Pluronic L64® are used as surfactant and encapsulating materials. Mircoencapsulation of quercetin by lyophilisation of SFEE-produced aqueous suspensions is also studied in order to compare it with the results of PGSS-drying. Samples are characterized considering encapsulation efficiency, antioxidant activity and Franz-cell measurements of transdermal diffusion of encapsulated quercetin product as assessment of its bioavailability. It is observed that samples encapsulated by SFEE and PGSS-drying are more homogeneous and less crystalline than samples obtained by lyophilisation, which results in a higher transdermal diffusivity.

Vitamin E in polycaprolactone nanoparticles was continuously produced by supercritical fluid extraction of emulsions using a high-pressure packing column in countercurrent mode. This operating mode reduces the amount of solvent required, increases production capacity and enables lower residual organic solvent concentrations in the raffinate. At 8.0 MPa and 313 K, with a packing height of 2 m, and a solvent to feed ratio of 5 kg L −1 , the residual acetone concentration was 1400 ppm, far below 5000 ppm, and therefore suitable for pharmaceutical applications. The process was also simulated with Aspen Plus. It would be necessary to increase the packing height to 3.5 m or the CO 2 flow rate to 60 g min −1 in order to get a residual acetone concentration suitable for food applications (50 ppm). The nanoparticles produced were non-aggregated spheres, which had an encapsulation efficiency higher than 70% and particle size at the nanoscale.

The encapsulation of vitamin E in polycaprolactone was done using four SFEE installations: A) a bubble column, B) a bubble column with gas redistributor, C) a spray column and D) a packed column. Performance of each installation was compared in terms of organic solvent removal, CO2 consumption and nanocapsule characteristics (encapsulation efficiency, particle size distribution and morphology). Bubble columns provided particles with good characteristics but required a high CO2 consumption and the production capacity were very low. The gas redistributor allowed decreasing the CO2 expenditure, but the operation was still discontinuous. Despite the high CO2 consumption in the spray column, the target acetone removal could not be achieved and particle morphology was negatively affected. The countercurrent operation in a packed column provided high production capacity, maintained good particle quality, and required a low amount of CO2 to remove the acetone.

α-Tocopherol (TOC) has become an important food additive due to its antioxidant effect and capacity to increase shelf-life of products. However, it is a hydrophobically active compound, which compromises its applicability in soft drinks and fruit beverages. The aim of this study was to develop sustained release forms of α-tocopherol using zein and cyclodextrin as carriers to be further applied in fruit beverage products for improved shelf-life. For this purpose, the encapsulation of α-tocopherol into zein protein was first optimized using a spray-drying technology. Different process conditions, namely temperature (110–180 °C) and solids concentration (0.01–0.04 g mL−1), were tested aiming at maximizing the encapsulation of α-tocopherol and antioxidant capacity using response surface methodology (RSM) following a Face Centred Design (FCD). Results showed that lower inlet gas temperature and lower solids concentration enable the production of α-tocopherol:zein forms with a higher antioxidant activity (oxygen radical absorbance capacity >90 μmol Trolox equivalent antioxidant capacity per g), and the highest encapsulation efficiency (42.05%) was obtained using 0.02 g mL−1 of solids concentration. β-Cyclodextrin was further added to the best system to increase the solubility capacity of α-tocopherol in fruit juice beverages. Despite the lower antioxidant activity (46.2 μmol Trolox equivalent antioxidant capacity per g), α-tocopherol:zein:cyclodextrin forms reduced colour losses when compared to the normal juice, and this effect was probably related to the suppression of anthocyanin degradation due to the formation of inclusion complexes with β-cyclodextrin. Importantly, none of these forms induced cytotoxicity in the Caco2 cell model, confirming the safety of these systems.

During the development of intranasal drug delivery systems for local/systemic effect or brain targeting, it is necessary to assess its cytotoxicity and drug transport through nasal epithelium. In order to avoid animal experiments or the use of excised tissues, in vitro cell models, such as RPMI 2650 cells, are being preferred during recent years. Nevertheless, the deposition of solid formulations into nasal cell layers with further transepithelial transport rate of drugs has been poorly studied or reported. Thus, the purpose of this work is to further investigate RPMI 2650 cell line as an effective alternative to animal tissues for solid drug-loaded formulations cytotoxicity and drug permeation studies in order to become an option as a tool for drug discovery. Furthermore, we wanted to determine the extent to which the administration of drugs in particulate forms would differ in relation to the permeability of the same compounds applied as solutions. RPMI 2650 cells were cultured in submersed or at air-liquid interface conditions and characterized regarding transepithelial electrical resistance (TEER) and production of mucus. Pure ketoprofen (used as model compound) and five formulations loaded with same drug, namely solid lipid particles (Gelucire 43/01™), structured lipid particles (Gelucire 43/01™:Glyceryl monooleate) and aerogel microparticles (Alginate, Alginate:Pectin, Alginate:Carrageenan), were evaluated with RPMI 2650 model in terms of cytotoxicity and permeability of drug (applied as solution, dispersion or powder+buffer). RPMI 2650 cells were capable to grow in monolayer and multilayer, showing the same permeability as excised human nasal mucosa for sodium fluorescein (paracellular marker), with analogous TEER values and production of mucus, as referred by other authors. None of the powders showed cytotoxicity when applied to RPMI 2650 cells. Regarding permeation of drug through cell layers, not only the form of application of powders but also their physical and chemical properties affected the final permeation of active pharmaceutical ingredient. Aerogel microparticles administered directly to the cell layer (powder+buffer) exhibited the highest permeation-enhancing effect compared to the pure drug, which can be attributed to the mucoadhesive properties of the materials composing the carriers, proving to be an attractive formulation for nasal drug delivery. According to these results, RPMI 2650 showed to be a promising alternative to ex vivo or in vivo nasal models for cytotoxicity and evaluation of drug permeability of nasal drug-loaded formulations.

Recent studies have shown that programmers' cognitive load during typical code development activities can be assessed using wearable and low intrusive devices that capture peripheral physiological responses driven by the autonomic nervous system. In particular, measures such as heart rate variability (HRV) and pupillography can be acquired by nonintrusive devices and provide accurate indication of programmers' cognitive load and attention level in code related tasks, which are known elements of human error that potentially lead to software faults. This paper presents an experimental study designed to evaluate the possibility of using HRV and pupillography together with eye tracking to identify and annotate specific code lines (or even finer grain lexical tokens) of the program under development (or under inspection) with information on the cognitive load of the programmer while dealing with such lines of code. The experimental data is discussed in the paper to assess different alternatives for using code annotations representing programmers' cognitive load while producing or reading code. In particular, we propose the use of biofeedback code highlighting techniques to provide online programmer's warnings for potentially problematic code lines that may need a second look at (to remove possible bugs), and biofeedback-driven software testing to optimize testing effort, focusing the tests on code areas with higher bug probability

This paper provides a study using Electroencephalography (EEG) to investigate the brain activity during code comprehension tasks. Three different code complexity levels according to five complexity metrics were considered. The use of EEG for this purpose is relevant, since the existing studies were mostly focused on neuroimaging techniques. Using Leave-One-Subject-Out cross-validation procedure for 30 subjects, it was found that the features related with the Gamma activity were the most common in all the folds. Regarding the brain regions, right parietal was the most frequent region contributing with more features. A Linear Discriminant Analysis Classifier for task classification, obtained a F-Measure of 92.71% for Code complexity easy, 52.25% for Code complexity intermediate and 53.13% for Code complexity advanced, revealing an evidence of mental effort saturation with the code complexity degree. This suggests that current code complexity metrics do not capture cognitive load and might not be the best approach to assess bug risk.

Our research explores a recent paradigm called Biofeedback Augmented Software Engineering (BASE) that introduces a strong new element in the software development process: the programmers' biofeedback. In this Practical Experience Report we present the results of an experiment to evaluate the possibility of using pupillography to gather biofeedback from the programmers. The idea is to use pupillography to get meta information about the programmers' cognitive and emotional states (stress, attention, mental effort level, cognitive overload,...) during code development to identify conditions that may precipitate programmers making bugs or bugs escaping human attention, and tag the corresponding code locations in the software under development to provide online warnings to the programmer or identify code snippets that will need more intensive testing. The experiments evaluate the use of pupillography as cognitive load predictor, compare the results with the mental effort perceived by programmers using NASATLX, and discuss different possibilities for the use of pupillography as biofeedback sensor in real software development scenarios.

This paper presents emergent experimental results showing that mental effort of programmers in code understanding tasks can be monitored through HRV (heart rate variability) using non-intrusive wearable devices. Results suggest that HRV is a good predictor for cognitive load when analyzing code and HRV results are consistent with the mental effort perceived by programmers using NASA-TLX. Furthermore, code complexity metrics do not correlate entirely with mental effort and do not seem a good indicator of the subjective perception of complexity felt by programmers. These first results are presented in the context of the project BASE-Biofeedback Augmented Software Engineering, which is briefly sketched, and proposes a radical neuroscience enabled approach to introduce biofeedback in software development.

Polymethoxylated flavones (PMFs) from citrus fruits are reported to present anticancer potential. However, there is a lack of information regarding their effect on cancer stem cell (CSC) populations, which has been recognized as responsible for tumor initiation, relapse, and chemoresistance. In this study, we evaluated the effect of an orange peel extract (OPE) and its main PMFs, namely, nobiletin, sinensetin, tangeretin, and scutellarein tetramethylether in targeting cell proliferation and stemness using a 3D cell model of colorectal cancer composed of HT29 cell spheroids cultured for 7 days in stirred conditions. Soft agar assay, ALDH1 activity, and relative quantitative gene expression analysis of specific biomarkers were carried out to characterize the stemness, self-renewal, and mesenchymal features of HT29 cell spheroids. Then, the impact of OPE and PMFs in reducing cell proliferation and modulating cancer stemness and self-renewal was assessed. Results showed that, when compared with monolayer cultures, HT29 cell spheroids presented higher ALDH1 activity (81.97% &plusmn, 5.27% compared to 63.55% &plusmn, 17.49% for 2D), upregulation of CD44, PROM1, SOX9, and SNAI1 genes (1.83 &plusmn, 0.34, 2.54 &plusmn, 0.51, 2.03 &plusmn, 0.15, and 6.12 &plusmn, 1.59 times) and high self-renewal capability (352 &plusmn, 55 colonies compared to 253 &plusmn, 42 for 2D). Incubation with OPE (1 mg/mL) significantly inhibited cell proliferation and modulated cancer stemness and self-renewal ability: colony formation, ALDH1 activity, and the expression of cancer stemness biomarkers PROM1 and LGR5 were significantly reduced (0.66 &plusmn, 0.15 and 0.51 &plusmn, 0.14 times, respectively). Among all PMFs, tangeretin was the most efficient in targeting the CSC population by decreasing colony formation and the expression of PROM1 and LGR5. Scutellarein tetramethylether was shown to modulate markers of mesenchymal/metastatic transition (increasing CDH1 and reducing ZEB1 and SNAI1) and nobiletin was capable of downregulating PROM1 and SNAI1 expression. Importantly, all PMFs and OPE were shown to synergistically interact with 5-fluorouracil, improving the antiproliferative response of this drug.

The application of biopolymer aerogels as drug delivery systems (DDS) has gained increased interest during the last decade since these structures have large surface area and accessible pores allowing for high drug loadings. Being biocompatible, biodegradable and presenting low toxicity, polysaccharide-based aerogels are an attractive carrier to be applied in pharmaceutical industry. Moreover, some polysaccharides (e.g. alginate and chitosan) present mucoadhesive properties, an important feature for mucosal drug delivery. This feature allows to extend the contact of DDS with biological membranes, thereby increasing the absorption of drugs through the mucosa. Alginate-based hybrid aerogels in the form of microparticles (

The efficiency of maltodextrin, whey protein isolate, and pea protein isolate to formulate a polyphenol-enriched grape marc extract by spray drying has been compared. Different inlet (120–140 °C ) and outlet (81–89 °C ) temperatures have been evaluated, as well as the amount of carrier expressed as carrier/extract ratio (0.1:1–1:1). The particles obtained were characterized in terms of their chemical composition, morphology, cytotoxicity, and cellular antioxidant activity. Outlet temperature was observed to have a higher influence on the particles than inlet temperature. A 22 % loss in total phenolic content was observed when the extract was spray dried without any carrier material, whereas the addition of the lowest amount of carrier lowered this value ( 87 %). Whey protein showed an enhancement of the chemical and cellular antioxidant activity per unit mass of gallic acid equivalent when compared to the other carriers.

Nanohydrogels were synthesized by microemulsion polymerization. These nanohydrogels were pre-designed to be pH and temperature stimuli-response materials, using N-isopropylacrilamide as a base monomer and 1-vinyl imidazole as ionizable comonomer. The pH sensitivity was confirmed by measuring the increase or decrease in volume in the nanoparticles by changing the pH of the medium. Nanoparticles were properly characterized by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) and transmission electron microscopy (TEM). Glass-transition temperature increased with vinyl imidazole content, and nanoparticles with average diameter of 68 nm were obtained. Particle size decreases with the increase in pH. After characterization, nanohydrogels were functionalized with folic acid to take advantage that the folate receptor is overexpressed in different types of cancer cells. The nanoparticles were loaded with the drugs: metformin, terfenadi...

Pea protein has been associated with promoting the satiety effect. One of the issues associated with the incorporation of pea protein in food products is the product homogeneity due to its solubility and dispersibility issues. Within this context, one goal of this study was to exploit the use of supercritical fluid technology to develop Solid Lipid Pea Particle (SLPP) aiming at improving dispersibility in fat-based products. PP was encapsulated by the PGSS® (Particles from Gas Saturated Solutions) technique into glyceryl dipalmitostearate (E471) and olive oil. Different process conditions, namely pressure (7.3–20.7 MPa), temperature (51–75 °C) and equilibrium time (3–37 min) were tested in order to optimize the encapsulation of pea protein via Response Surface Methodology (RSM), following a Central Composite Rotatable Design (CCRD). Results showed that pressure and the interaction between pressure and temperature had a significant impact (p < 0.05) on the protein load and thus on the encapsulation efficiency. The highest encapsulation efficiency (96%) was achieved at 14 MPa, 51 °C and 20 min. Under these conditions, SLPP presented 0.15 mg of protein per mg of particles and 84% of lipase inhibitory activity. When compared with the PP (non-encapsulated), liposoluble pea protein particles contributed to a better product homogenization. The food industry can also take advantage of the ability of pea protein for foam stabilization in aqueous food products. Therefore, PP was treated with high-pressure supercritical CO2 treatment (HPT-scCO2) that has led to improved foaming properties when compared with the non-treated PP.

In this work, pressurized fluid extraction was explored to recover isothiocyanates (ITCs) and phenolic compounds from watercress. Pretreatment of the raw material was studied, using different conditions of temperature (25 °C; 35 °C), pressure (Patm; 25 MPa), incubation time (0; 30; 60; 120 min) and moisture content (125; 250; 900% of water, dry basis), aiming at promoting the enzymatic hydrolysis of glucosinolates in ITCs. Extractions of ITCs were performed with supercritical CO2, and different mixtures of CO2 : ethanol (0–50, % w/w) were applied to obtain phenolic-enriched ITCs extracts. Extractions were performed at 35 °C and 25 MPa for 2 h and extracts analyzed concerning total ITCs, phenethyl isothiocyanate (PEITC) content, phenolic composition, antioxidant capacity (using ORAC, HORAC and HOSC assays) and antiproliferative effect in a human colorectal cancer cell line (HT-29). Results showed that supercritical CO2 was highly selective in isolating ITCs from watercress (up to 31.7 ± 1.6 μmol ITC per g). When mixtures of CO2 : ethanol were used, extraction of phenolics (up to 10.1 ± 0.8 mg GAE per g) and antioxidants (up to 204.4 ± 21.5 μmol TE per g concerning ORAC, 70.8 ± 10.7 μmol CAE per g for HORAC and 189.5 ± 22.9 μmol TE per g for HOSC), was promoted. PEITC was the main compound responsible for the inhibition of cancer cell growth of all extracts (EC5024 h = 27.8 ± 1.9 μM PEITC). However, extracts obtained by supercritical CO2 extraction after a 30 minute incubation period with CO2 and with CO2 : ethanol mixtures (80 : 20 and 60 : 40, % w/w) were revealed to be effective in isolating other bioactive compounds that enhanced the antiproliferative response of extracts (EC5024 h values of 23.1 ± 0.9; 20.7 ± 1.9 and 19.8 ± 0.7 μM PEITC, respectively).

Epigallocatechin gallate (EGCG) is the catechin with the highest antioxidant activity present in green tea. Nevertheless, due its low bioavailability, it is necessary to develop EGCG formulations capable of improving its stability resulting in increased bioavailability and thus higher biological activity of this catechin (e.g. antioxidant activity). The purpose of this work was the formulation of EGCG using three distinct natural origin carriers, namely OSA-starch, soybean lecithin and β-glucan, by particles from gas saturated solution drying (PGSS-drying). Non-cytotoxic solid formulations of EGCG in the range of micrometers and encapsulation efficiencies up to 80.5% were obtained. An improved antioxidant activity (AA) determined by the oxygen radical absorbance capacity (ORAC) method was obtained for all formulations. Furthermore, lecithin:EGCG and β-glucan:EGCG presented higher cellular antioxidant activity (Caco-2 cells) values than free EGCG at the same concentrations tested (around 1.5-fold higher values). Moreover, the solid formulations presented preservation of the AA over 4 months, and an improved storage stability in comparison with non-encapsulated EGCG over 48 h at 328 K in the absence of light (accelerated storage). These results show that PGSS-drying conditions enabled the preservation of the bioactive properties of catechin, allowing the formulation of solid particles with enhanced features.

The aim of this work was to investigate and compare the phenolic profile of 15 wild growing blackthorn (Prunus spinosa L.) genotypes from the slopes of Fruska Gora mountain in north Serbia. Their effect in inhibiting i) α-amylase and α-glucosidase activities and ii) colorectal cancer cell line (HT29) growth was also studied. Blackthorn fruit extracts exhibited high phenolic content being enrich in anthocyanins. Principal component analysis was used to correlate the bioactive response with phenolic composition. It was found that derivatives quercetin and anthocyanin peonidin are the major contributors of the inhibition of carbohydrates hydrolyzing enzymes as well as with the antiproliferative effect of blackthorn. Among all samples, the genotype from Beska locality showed the higher capacity in inhibiting alpha-amylase, alpha-glucosidase and HT29 cell growth. Because of high anthocyanin content and higher bioactive response, these genotypes could be recommended for the further cultivation and investigation.

Nanoemulsions can be used to improve the bioaccessibility of lipophilic bioactive compounds, such as curcumin, being their behaviour highly influenced by their interfacial properties. The effect of chitosan and alginate layers deposition on curcumin nanoemulsions behaviour during in vitro digestion was evaluated using a dynamic gastrointestinal system. Results showed that polyelectrolyte layers deposition improved curcumin antioxidant capacity during in vitro digestion. In addition, multilayer nanoemulsions showed a better control of the rate and extent of lipid digestibility by decreasing free fatty acids release, compared to uncoated nanoemulsions. However, a lower curcumin bioaccessibility was observed for multilayer nanoemulsions. Although cytotoxicity assays revealed that both nanosystems are toxic due to the use of sodium dodecyl sulphate (SDS), nanosystems were 3.3-fold less toxic than SDS itself. This study showed that multilayer nanoemulsions could be used to increase satiety by retarding lipid digestion, which can be important for functional foods development for combating obesity., The authors Hélder D. Silva, Ana C. Pinheiro and Miguel A. Cerqueira (SFRH/BD/81288/2011, SFRH/BPD/101181/2014 and SFRH/BPD/72753/2010, respectively)aretherecipientsofafellowship from the Fundação para a Ciência e Tecnologia (FCT, Portugal). This work was supported by Portuguese Foundation for Science and Technology (FCT) under the scope of the Project PTDC/AGR-TEC/5215/2014, of the strategic funding of UID/BIO/04469/2013 Unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte, PEst-OE/EQB/LA0004/2011 and iNOVA4Health – UID/Multi/04462/2013, a program financially supported by FCT/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. The authors also acknowledge the European Commission: BIOCAPS (316265, FP7/REGPOT-2012-2013.1). This work was also supported by the “CARINA” project for the safeness, sustainability and competitiveness of agro-food productions of Campania Region. The support of EU Cost Action FA1001 is gratefully acknowledged. The authors also acknowledge Stepan (The Netherlands) for providing the Neobee 1053 oil, and Rui Fernandes from IBMC, University of Porto for assistance in taking the TEM pictures., info:eu-repo/semantics/publishedVersion

Polymethoxylated flavones (PMFs) have been recognized to inhibit colorectal cancer proliferation through various mechanisms, however most of these studies have been performed on cells grown as monolayers that present limitations in mimicking the 3D tumor architecture and microenvironment. The main aim of this study was to investigate the anticancer potential of an orange peel extract (OPE) enriched in PMFs in a 3D cell model of colorectal cancer. The OPE was developed by supercritical fluid extraction and the anticancer effect was evaluated in HT29 spheroids cultures in a stirred-tank based system.Results showed that OPE inhibited cell proliferation, induced cell cycle arrest (G2/M phase), promoted apoptosis, and reduced ALDH+ population on HT29 spheroids. The antiproliferative activity was significantly lower than that obtained for 2D model (EC50 value of 0.43 ± 0.02 mg/mL) and this effect was dependent on diameter and cell composition/phenotype of spheroids derived from different culture days (d...

Production of lipid particles loaded with metal nanoparticles by supercritical fluids based processes has been barely studied. In this work, copper nanoparticles were loaded into glyceryl palmitostearate microparticles by PGSS® (Particles from Gas Saturated Solutions). The effect of different variables, temperature (60-80 ºC), copper load (0.2-5%w/w) and water addition (0 – 40%w/w), in particle size and encapsulation efficiency has been studied. The dispersion of metal nanoparticles in the lipid has been determined by SEM-FIB coupled with EDS mapping. In all cases, mean particle size values lower than 70 μm have been obtained, and encapsulation efficiencies around 60% have been achieved. The addition of water has no negative effect in encapsulation efficiency nor in nanoparticles dispersion within the lipid microparticle, being important since nanoparticles are commonly synthetized in aqueous medium., 2020-01-10, Ministerio de Ciencia e Innovación and the University of Valladolid (JCI-2012-14992), Fundação para a Ciência e a Tecnologia (FCT) (Grant PEst-OE/EQB/LA0004/2011: SFRH/BD/77350/2011), Fundação para a Ciência e a Tecnologia (FCT) and FEDER 2014-2020 iNOVA4Health – UID/Multi/04462/2013 and UID/Multi/04551/2013 (GreenIT)

Aging wine lees are water-wastes produced during the wine aging inside wood barrels that can be considered as alternative sources of bioactive compounds. Phenolic characterization and antioxidant activity (AA) measurements of wines lees solid-liquid extracts have been undertaken on a dry extract (DE) basis. Solvents with different polarities (water, methanol, ethanol, two hydroalcoholic mixtures and acetone) were used. Total phenolic (TPC) and total flavonoid contents (TFC) were determined. The mixture of 75:25(v/v) EtOH:H2O showed the highest values with 254mgGAE/gDE and 146mgCATE/gDE respectively. HORAC, HOSC and FRAP were used to determine the AA of the extracts being also highest for the mixture of 75:25(v/v) EtOH:H2O (4,690 µmolCAE/gDE, 4,527 µmolTE/gDE and 2,197 µmolTE/gDE, respectively). For ORAC method, methanol extract showed the best value with 2,771µmolTE /gDE. Correlations between TPC, TFC, phenolic compounds and AA were determined. Most relevant compounds contributing to AA were identified using data from mass spectrometry, being mainly anthocyanins., 2019-04-01

Matricaria Pubescens (Asteraceae) is a well known medicinal plant used in the south west of Algeria against several diseases. This study was devoted to the determination of polyphenols, flavonoids, and condensed tannins contents of M . pubescens after their extraction using various solvents with different polarities (distilled water, aqueous acetone (50%), aqueous methanol (50%), aqueous ethanol (50%), acetone (100%), methanol (100%), and ethanol (100%)), as well as the determination of the antioxidant and the antibacterial activities of all obtained extracts. The results obtained indicate that the aqueous methanol (50%) and the aqueous ethanol (50%) extracts presented the highest contents of polyphenols. The antioxidant activity was evaluated using ferric reducing power, DPPH radical scavenging activity, ABTS radical scavenging activity, oxygen radical absorbance capacity (ORAC) and hydroxyl radical scavenging capacity (HOSC). The results showed that all extracts of M . pubescens, prepared using different solvent, have diverse antioxidant capacities, however aqueous methanol (50%), aqueous ethanol (50%) and aqueous acetone (50%) exhibited the highest antioxidant capacity. Extracts of M . pubescens exhibited an inhibiting capacity against the tested strains except: Escherichia coli ATCC 25922 and Acinetobacter baumannii 897. The aqueous methanol (50%) and the aqueous ethanol (50%) extracts were most effective; the greatest zone of inhibition was obtained with the aqueous ethanol (50%) extract against Methicillin-resistant Staphylococcus aureuscStaphylococcus aureusc . The minimum inhibitory concentration (MIC) with the value of 0.78 mg/mL obtained against A. baumannii 610 using the aqueous methanol (50%) extract indicate that this plant is a potential natural antimicrobial agent. These biological activities could support the traditional use of M . pubescens .

Cactus pears (Opuntia spp.) have been identified as a promising betalainic crop covering a wide colour spectrum from yellow to purple pigments. The aim of this study was to exploit the use of supercritical fluid technology to develop lipidic particles of betalain-rich extract derived from Opuntia spp. fruits to be further incorporated in food matrices. A natural extract was encapsulated by the PGSS® (Particles from Gas Saturated Solutions) technique into glyceryl monoostearate, using a surfactant (polyglyceryl-3 polyricinoleate) and water. Different process conditions, namely pressure (9–23 MPa), temperature (57–73 °C) and equilibrium time (5–55 min), were tested in order to model the encapsulation of betalains via the Response Surface Methodology (RSM), following a Central Composite Rotatable Design (CCRD). Results showed that temperature and pressure were the parameters that mostly affected the encapsulation of the natural pigment and the highest betacyanin encapsulation was achieved at lower pressures. This effect was more pronounced at higher temperatures and lower equilibrium times. Under these conditions, Opuntia spp. particles presented 64.4 mg per 100 g of betacyanins and high antioxidant capacity. In addition, the particles with higher betacyanin content showed an average particle size of 10 μm and a melting point between 57 and 65 °C. When compared with the Opuntia dried extract, lipidic particles contributed to a better homogenization of the pink colour after incorporation in ice cream.

Hybrid lipid–polymer particles are gaining increasing interest to be applied as drug delivery systems due to their greater stability in biological fluids and enhanced cellular uptake of drugs. However, a major drawback is the fact that these particles are usually produced through techniques that use organic solvents. This work studies the possibility of producing for the first time hybrid particles composed by lipid multicores enveloped in a polymeric layer through Particles from Gas Saturated Solutions (PGSS ® ), without using organic solvents. An oil-in-water emulsion, composed by Gelucire 43/01™ (GEL) as the discontinuous phase and by an aqueous polyethylene glycol 4000 (PEG) solution as the continuous phase, was successfully precipitated by PGSS ® . Operating conditions that ensured the stability of the CO 2 saturated emulsion were previously evaluated. The resulting PEG–GEL particles present a spherical-like morphology constituted by several lipid cores encapsulated into a polymeric shell. The applicability of these structured particles to be used as drug delivery system (DDS) was studied by using ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID), as model drug. The particles provided an initial burst release of the drug due to the progressive dissolution of the external layer of PEG, followed by a controlled release of the NSAID from the GEL cores.

Cactus pears (Opuntia spp.) have been identified to be an excellent source of betalain pigments which can be used as a red food colourant. In this work, pigments from cactus pear fruits were effectively extracted by high pressure carbon dioxide assisted-acidified water extraction after a pre-treatment with CO2 at 375 bar, 55 °C and 60 minutes. Different process conditions, namely pressure, temperature and volume ratio of (solid–liquid mixture)/(pressurized CO2), were tested in order to model the extraction of betalains from prickly pears via the response surface methodology. The best response was achieved at 100 bar, 40 °C and 20% volume ratio of (solid–liquid mixture)/(pressurized CO2). Under these conditions, the betalains yield was 89 ± 0.7 mg per 100 g of dried fruit, 83% of the maximum extractable pigments from Opuntia spp. fruits. Furthermore, Opuntia spp. extracts presented a vivid red colour with high antioxidant activity.

Colorectal cancer (CRC) recurrence is often attributable to circulating tumor cells and/or cancer stem cells (CSCs) that resist to conventional therapies and foster tumor progression. Isothiocyanates (ITCs) derived from Brassicaceae vegetables have demonstrated anticancer effects in CRC, however little is known about their effect in CSCs and tumor initiation properties. Here we examined the effect of ITCs-enriched Brassicaceae extracts derived from watercress and broccoli in cell proliferation, CSC phenotype and metastasis using a previously developed three-dimensional HT29 cell model with CSC-like traits. Both extracts were phytochemically characterized and their antiproliferative effect in HT29 monolayers was explored. Next, we performed cell proliferation assays and flow cytometry analysis in HT29 spheroids treated with watercress and broccoli extracts and respective main ITCs, phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). Soft agar assays and relative quantitative expression analysis of stemness markers and Wnt/β-catenin signaling players were performed to evaluate the effect of these phytochemicals in stemness and metastasis. Our results showed that both Brassicaceae extracts and ITCs exert antiproliferative effects in HT29 spheroids, arresting cell cycle at G2/M, possibly due to ITC-induced DNA damage. Colony formation and expression of LGR5 and CD133 cancer stemness markers were significantly reduced. Only watercress extract and PEITC decreased ALDH1 activity in a dose-dependent manner, as well as β-catenin expression. Our research provides new insights on CRC therapy using ITC-enriched Brassicaceae extracts, specially watercress extract, to target CSCs and circulating tumor cells by impairing cell proliferation, ALDH1-mediated chemo-resistance, anoikis evasion, self-renewal and metastatic potential.

The authors thank the fellowships (SFRH/BPD/72753/2010, SFRH/BPD/101181/2014, and SFRH/BDE/51856/2012) from the Fundacao para a Ciencia e Tecnologia (FCT, Portugal). The authors also thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the project “BioInd—Biotechnology and Bioengineering for improved Industrial and Agro-Food processes,” REF. NORTE07-0124-FEDER-000028 Cofunded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER, CNPq (Brazil).

Producción Científica, Microwave assisted extraction advantages are widely recognised. However, its implementa-tion at industrial scale is restricted due to microwave limitations. In this work, a microwavepretreatment is proposed as an easy scale-up alternative for grape pomace polyphenolextraction, especially for anthocyanins. The double effect of this pretreatment on extractionyield and on product richness is assessed. Microwaves accelerate the extraction kineticsof most compounds, but their effect on polyphenols is more pronounced than in othersubstances (like sugars and fibres). These differentiated rates are exploited to improve thepolyphenol richness of the final dry product. By selecting the appropriate operating condi-tions, polyphenol yield was increased by 57% and, simultaneously, dry product richness wasenhanced by 32%. Also, anthocyanin extraction boost was remarkable. Its content in the finaldry product was 85% higher than the one obtained without the microwave pretreatment.The cellular bioactivity of these extracts was improved by 83% and 133%.

The main effects of three different irrigation regimes, i.e., sustained deficit irrigation (SDI), regulated deficit irrigation (RDI) and non-irrigated (NI), on seed traits namely proanthocyanidins (PAs) were evaluated in the wine grape cultivar Aragonez (syn. Tempranillo) grown in Alentejo (Portugal) over two growing seasons. Results showed that while the number of seeds per berry was not affected by water availability, seed fresh weight differed among treatments, the NI treatment exhibiting the lowest values. The biosynthetic pathway of flavanols appeared to be modified by the irrigation treatment, and several genes responsible for PA synthesis were up-regulated in the most stressed seeds (RDI and NI). However, this effect had no impact on PA content, suggesting the influence of other factors such as oxidation and/or degradation of PAs at late stages of maturation in grape seeds. The seeds’ non-enzymatic antioxidant capacities (oxygen radical absorbance capacity (ORAC) and hydroxyl radical adverting capacity (HORAC)) were modulated by water deficit and correlated well with PA content. The impact of irrigation strategy on PA biosynthesis, content, and anti-radical activity during seed ripening is discussed in the context of increasing interest in the role of PAs in the color and taste of wine, and the potential health benefits relating to their antioxidant capacity.

In this work, Opuntia ficus indica juice was explored as a potential source of natural antioxidant and anti-inflammatory ingredients towards intestinal inflammation. An adsorption separation process was used to produce a natural flavonoid-rich concentrate (FRC) from Opuntia ficus-indica juice. The FRC effect (co- or pre-incubation) on induced-oxidative stress and induced-inflammation was evaluated in human Caco-2 cells. The main constituents identified and present in the extract are flavonoids (namely isorhamnetins and their derivatives such as isorhamnetin 3-O-rhamnose-rutinoside and isorhamnetin 3-O-rutinoside) and phenolic acids (such as ferulic, piscidic and eucomic acids). Our results showed that co-incubation of FRC with the stress-inducer attenuates radicals production in a much more significant manner than pre-incubation. These results suggest that FRC compounds which cannot pass the cell membrane freely (isorhamnetin derivatives) have an ability to inhibit the formation of H2O2-induced radicals in the surrounding environment of intestinal epithelial cells. The capacity of FRC (co-incubation) for suppressing (at the extracellular level) free radicals chain initiation or propagation reaction was probably related with a more pronounced reduction in protein oxidation. A similar response was observed in the inflammatory state, where a marked decrease in IL-8 secretion and blocked degradation of IκBα was achieved for FRC co-incubation. Simultaneously, treatment with FRC significantly reduces NO and TNF-α expression and modulates apparent permeability in Caco-2 cells. In these cases, no significant differences were found between pre- and co-incubation treatments suggesting that bioavailable phenolics, such as ferulic, eucomic and piscidic acids and isorhamnetin, act at the intracellular environment.

Berries are an important dietary source of fibres, vitamins, minerals and some biologically active non-nutrients. A red raspberry fruit extract was characterized in terms of phenolic content and the anti-inflammatory properties and protective effects were evaluated in two experimental models of inflammation. The antioxidant potential of the extract, the cellular antioxidant activity and the effects over neutrophils' oxidative burst were also studied to provide a mechanistic insight for the anti-inflammatory effects observed. The extract was administered in a dose of 15 mg kg(-1), i.p. and significantly inhibited paw oedema formation in the rat. The same dose was administered via i.p. and p.o. routes in the collagen-induced arthritis model in the rat. The extract showed pharmacological activity and was able to significantly reduce the development of clinical signs of arthritis and markedly reduce the degree of bone resorption, soft tissue swelling and osteophyte formation, preventing articular destruction in treated animals.

The market for natural bioactive ingredients is one of the most attractive at the moment, and natural lipophilic compounds of various structures and functionalities provide excellent molecules for the production of nutraceuticals, functional foods, and food additives. The use of these products in various commercial sectors and the consumer demand for “natural”, together with increasingly strict environmental regulation related to the use of organic solvents, particularly with products for human consumption, have created a great challenge for the natural ingredients industry. In order to address the need for safer and healthier foods and to offer competitive products aimed at meeting consumer expectations, many scientists and manufacturers are making genuine efforts to explore new “green sustainable processes” for obtaining bioactive ingredients. In this chapter, both conventional and new alternative techniques for the isolation of high-value lipophilic bioactives from different natural matrixes are presented. One new technology, supercritical fluid extraction (SCFE), is reviewed in greater detail. The practical issues associated with each extraction method are also discussed, as well as the potential for upscaling of the technology.

During the last years, special interest has been focused on the recovery of bioactive compounds from food industry wastes in an effort to find added value applications for residues. In this work, Opuntia spp. residues from fruit juice production were explored as potential sources of natural chemotherapeutic ingredients towards colon cancer. Hydroalcoholic extraction and adsorption separation processes (with Amberlite XAD16 resin) were used to produce natural extracts from residues of Opuntia ficus-indica and Opuntia robusta and their antiproliferative effect was evaluated in human colon carcinoma HT29 cell line. Our results showed that the natural extracts efficiently inhibited cancer cell growth and induced cell cycle arrest in different checkpoints—G1, G2/M and S. The phytochemical constituents presented in the samples, namely betacyanins, flavonoids (isorhamnetin derivatives) and phenolic acids (ferulic acid) were identified as the main responsible compounds for the cell cycle arrest. Moreover, the extracts that presented the lowest effective dose values in terms of polyphenols (ED50 value

For the first time, gas-filled microparticles were successfully prepared using a supercritical fluid based technology. Low molecular weight perfluorcarbon (PFC) gases, C3F8 or C4F8, have been encapsulated into Gelucire® 50/13 (lipid-based carrier: polyethylene glycol glycerides), using PGSS® (Particles from Gas Saturated Solution) technique. Particles were produced from the fast expansion of the melted lipid carrier saturated with a mixture of (CO2 + PFC). The presence of the gas into the produced microparticles was verified by Nuclear Magnetic Resonance (NMR) analysis of fluorine atom. The effect of carrier to PFC mass ratio and PFC structure on the entrapment efficiency of the PFC gas into the particles was evaluated at fixed at 8.5 MPa and 353 K. These parameters were fixed in a preliminary study according to the morphology, size and flowability of the particles. The stability of encapsulated C4F8 in microparticles showed to be higher than C3F8; it was determined to be 2 h, at room conditions at the optimized carrier: PFC mass ratio of 30:1.

Oregano (Origanum vulgare) essential oil (EO), besides its flavoring characteristics, is well known for its antioxidant, antimicrobial and anti-inflammatory properties, which make it a good alternative as food preservative from natural origin. Furthermore, it has been reported that oregano EO can be used as a health promoting substance in the prevention of neurodegenerative disorders. However, EO can be easily degraded by light, oxygen and temperature and there is still a lack of an adequate protection system to improve its stability along processing. In the present study, oregano essential oil was impregnated in microspheres of different types of starch using SSI (supercritical solvent impregnation) with supercritical carbon dioxide (sc-CO2). This process is characterized by mild operating conditions and inert processing atmosphere, especially adequate for the formulation of EOs. The quality of the final product and process efficiency was evaluated by the antioxidant activity (AA) of the final product, determined according to the ORAC assay. Different processing conditions were studied, such as pressure (8–15 MPa), temperature (40–50 °C), impregnation time (3–24 h) and mass ratio of essential oil/starch (0.4–3). The SSI is a batch process and the influence of mode of depressurization was also analyzed. Industrial relevance The present study demonstrates that supercritical impregnation is an attractive process for the impregnation of natural matrices, such as starch granules, with essential oils (EOs), namely oregano essential oil. The mild operating conditions avoid EO degradation and the high diffusivity of supercritical CO2 in the solid matrix (starch) guarantees a deep impregnation of the EO and high antioxidant activity of the product, also during storage. Further, the process is easy to scale up for the production of potential health-promoting ingredients for the food industry.

The formulation of S-(+)-ibuprofen as a model water-insoluble drug in different carrier materials (poloxamers, gelucire and glyceryl monostearate, GMS) by Particles from Gas Saturated Solutions (PGSS) technique has been studied. Porous, spherical particles of 50⿿200 μm have been obtained with encapsulation efficiencies up to 90%. Differential scanning calorimetry assays reveal modifications on the structure of the material, with formation of a solid solution in experiments with poloxamer carriers, and formation of solid dispersions with a possible reduction of the crystallinity of the carrier in experiments with GMS. Drug release tests in simulated gastric and intestinal fluids have been performed. Formulations with poloxamer carrier materials provided an increased solubility of ibuprofen in the gastrointestinal fluids, with a very fast release and dissolution of this compound, while gelucire and GMS carriers did not improve the solubility of ibuprofen, but provided a slower, controlled release of the drug. PGSS-processed samples presented a superior performance over physical mixtures in terms of the solubility increase and the control of the release rate. These results show the wide possibilities and flexibility of the PGSS technique for the development of hybrid formulations of water-insoluble active compounds with hydrophilic or hydrophobic carrier materials, achieving either an increased, accelerated dissolution, or a slower, controlled delivery, depending on the choice of carrier materials.

Lavandin essential oil has been encapsulated in poly-(ϵ-caprolactone) (MW: 4000 g mol–1) by means of the particles from gas saturated solutions (PGSS) process. The influence of process conditions, i.e., pre-expansion temperature and pressure, and oil/polymer mass ratio, on the morphology of particles and the efficiency of encapsulation has been analyzed. Spherical particles with particle sizes of 100–700 μm were obtained, with increasing particle sizes and more agglomeration as the oil/polymer ratio was increased. The efficiency of encapsulation was increased by conditions that favored a fast solidification of the polymer shell, including high pre-expansion pressures, reaching efficiencies up to 50 % with oil loads up to 120 mg oil/g particles.

Composites of amoxicillin and ethyl cellulose in the micrometer range were precipitated by supercritical antisolvent (SAS) processes using carbon dioxide as antisolvent and a mixture of dichloromethane and dimethylsulfoxide as solvents. Morphologies and mean diameter ranges were analyzed by scanning electron microscopy. X-ray photoelectron spectroscopy (XPS) and high-performance liquid chromatography were carried out in order to ensure successful coprecipitation and to determine the amoxicillin contents in the final products. The SAS processes used differ mainly in the way in which the solutions are introduced: through a normal or a coaxial nozzle. The XPS results provided proof that amoxicillin was not distributed in the same way in all the samples. The release behavior of the composites obtained was evaluated in two biological fluids, i.e., simulated gastric and simulated intestinal fluids. The different systems led to the release of the drug in different ways; but in all cases slower solubilization was obtained than for the pure drug.

Lavandin (Lavandula hybrida) essential oil contains components with biocide and antiviral properties that can be used as substitutes of antibiotics. This application requires an appropriate formulation of the essential oil. In the present work, the antimicrobial activity of free and encapsulated lavandin essential oil against three pathogenic bacteria (Gram-negative: Escherichia coli; Gram-positive: Staphylococcus aureus and Bacillus cereus) was determined. The formulations were prepared using innovative high-pressure techniques (PGSS and PGSS-drying) as well as spray-drying. Carrier materials used for the encapsulation were soybean lecithin, n-octenyl succinic anhydride (OSA) modified starch and poly-caprolactone. Results demonstrated that lavandin oil antibacterial activity could be enhanced by encapsulation, due to the protection and control release of the oil. As well, encapsulation might present an interesting opportunity to facilitate the action of antimicrobials, improving essential oil penetration inside of the outer membrane.