Method to prepare aqueous propolis dispersions based on phase separation.

Propolis has many benefits for human health. To facilitate its oral consumption, we designed propolis-in-water dispersions to be used as nutraceuticals. Propolis was first dissolved either in ethanol or in a hydroalcoholic solution. Water being a non-solvent for propolis, its addition produced propolis precipitation. We explored the ternary phase diagram of water, propolis and ethanol to identify the line separating the one phase region where propolis is fully dissolved, and the two-phase region where a concentrated propolis solution coexists with a dilute one. Droplets rich in propolis were produced during the phase separation process under mechanical stirring induced by a rotor-stator device or a microfluidizer, and they were stabilized using gum Arabic as an emulsifier. Ethanol was finally removed by distillation under reduced pressure. Propolis dispersions in the micron and submicron size range could be obtained. They contained between 1.75 and 10.5 wt% polyphenols relative to the total mass.

Early stimulation of the left posterior parietal cortex promotes representation change in problem solving.

When you suddenly understand how to solve a problem through an original and efficient strategy, you experience the so-called "Eureka" effect. The appearance of insight usually occurs after setting the problem aside for a brief period of time (i.e. incubation), thereby promoting unconscious and novel associations on problem-related representations leading to a new and efficient solving strategy. The left posterior parietal cortex (lPPC) has been showed to support insight in problem solving, when this region is activated during the initial representations of the task. The PPC is further activated during the next incubation period when the mind starts to wander. The aim of this study was to investigate whether stimulating the lPPC, either during the initial training on the problem or the incubation period, might enhance representation change in problem solving. To address this question, participants performed the Number Reduction Task (NRT, convergent problem-solving), while excitatory or sham (placebo) transcranial direct current stimulation (tDCS) was applied over the lPPC. The stimulation was delivered either during the initial problem representation or during the subsequent incubation period. Impressively, almost all participants (94%) with excitatory tDCS during the initial training gained representational change in problem solving, compared to only 39% in the incubation period and 33% in the sham groups. We conclude that the lPPC plays a role during the initial problem representation, which may be considerably strengthened by means of short brain stimulation.

Hydroxytyrosol encapsulated in biocompatible water-in-oil microemulsions: How the structure affects in vitro absorption.

Over the last years, the incorporation of natural antioxidants in food and pharmaceutical formulations has gained attention, delaying or preventing oxidation phenomena in the final products. In order to take full advantage of their properties, protection in special microenvironments is of great importance. The unique features of the natural phenolic compound hydroxytyrosol (HT) - including antioxidant, anti-inflammatory, antiproliferative and cardioprotective properties - have been studied to clarify its mechanism of action. In the present study novel biocompatible water-in-oil (W/O) microemulsions were developed as hosts for HT and subsequently examined for their absorption profile following their oral uptake. The absorption of HT in solution was compared with the encapsulated one in vitro, using a coculture model (Caco-2/TC7 and HT29-MTX cell lines). The systems were structurally characterized by means of Dynamic Light Scattering (DLS) and Electron Paramagnetic Resonance (EPR) techniques. The diameter of the micelles remained unaltered after the incorporation of 678 ppm of HT but the interfacial properties were slightly affected, indicating the involvement of the HT molecules in the surfactant monolayer. EPR was used towards a lipophilic stable free radial, namely galvinoxyl, indicating a high scavenging activity of the systems and encapsulated HT. Finally, after the biocompatibility study of the microemulsions the intestinal absorption of the encapsulated HT was compared with its aqueous solution in vitro. The higher the surfactants' concentration in the system the lower the HT concentration that penetrated the constructed epithelium, indicating the involvement of the amphiphiles in the antioxidant's absorption and its entrapment in the mucus layer.

Emulsification of non-aqueous foams stabilized by fat crystals: Towards novel air-in-oil-in-water food colloids.

We designed Air-in-Oil-in-Water (A/O/W) emulsions. First, Air-in-Oil foams were fabricated by whipping anhydrous milk fat. The maximum overrun was obtained at 20 °C. The foams contained 30-35 vol% air and were stabilized solely by fat crystals. To refine the bubble size, foams were further sheared in a Couette's cell. The average bubble size reached a value as small as 6.5 µm at a shear rate of 5250 s-1. The nonaqueous foams were then dispersed in a viscous aqueous phase containing sodium caseinate to obtain A/O/W emulsions. The shear rate was varied from 1000 to 7500 s-1, allowing to obtain Air-in-Oil globules whose average diameter ranged from 15 to 60 µm. To avoid globule creaming, the aqueous phase was gelled by incorporating hydroxyethyl cellulose. Homogeneous emulsions were obtained with fat globules containing around 22 vol% of residual air. The systems were kinetically stable for at least 3 weeks at 4 °C.

O/W Pickering emulsions stabilized by cocoa powder: Role of the emulsification process and of composition parameters.

We fabricated oil-in-water emulsions stabilized by delipidated commercial cocoa powder. The emulsions were characterized in terms of droplets and particles size distribution and interfacial coverage by cocoa powder by developing methods to separate droplets from adsorbed and unadsorbed cocoa particles. Three different processes were compared for their ability to produce fine and stable emulsions: rotor/stator turbulent mixing, sonication and microfluidization. Among those techniques, microfluidization was the most performing one. In this case, micron-sized emulsions with narrow size distributions could be obtained with >90 wt% of the powder insoluble material anchored to the interfaces, and they were still stable after 90 days. It was demonstrated that the mixing process did not generate finer cocoa particles but provoked disentanglement of the large primary particles, providing them an open, expanded structure that facilitated emulsification. It was also shown that the finer insoluble fraction of the powder and the soluble fraction had no significant impact on emulsification and on kinetic stability. In the poor particles regime, the oil-water interfacial area varied linearly with the amount of adsorbed powder, suggesting that the final droplet size was controlled by the so-called limited coalescence process, as already observed in conventional Pickering emulsions stabilized by spherical solid particles.

Monodisperse Oil-in-Water Emulsions Stabilized by Proteins: How To Master the Average Droplet Size and Stability, While Minimizing the Amount of Proteins.

Hexadecane-in-water emulsions were fabricated by means of a microfluidizer using two types of protein stabilizers, sodium caseinate (NaCAS) and ß-lactoglobulin (BLG). A study of the dependence of the mean droplet diameter and protein coverage on protein concentration was performed. At low protein concentrations, the emulsions were monodispersed and their mean droplet size was governed by the so-called limited-coalescence process. In this regime, the interfacial coverage was constant and was deduced from the linear evolution of the total interfacial area as a function of the amount of adsorbed proteins. In emulsions based on NaCAS, almost all of the initial protein contents were adsorbed at the interfaces. Emulsions formulated at very low protein content underwent unlimited coalescence after prolonged storage or when submitted to centrifugation. Additional NaCAS was incorporated in the continuous phase, right after the emulsification process, as a means of ensuring kinetic stability. The interfacial coverage increased after protein addition. Other strategies including acidification and salt addition were also probed to gain stability. Instead, in emulsions based on BLG, only partial adsorption of the initial protein content was observed. The corresponding emulsions remained kinetically stable against coalescence, and no further addition of protein was required after emulsification. Our approach allows to obtain monodisperse, kinetically stable emulsions and to master their average droplet size, while minimizing the amount of proteins.

Microemulsion versus emulsion as effective carrier of hydroxytyrosol.

Two edible Water-in-Oil (W/O) dispersions, an emulsion that remains kinetically stable and a microemulsion which is spontaneously formed, transparent and thermodynamically stable, were developed for potential use as functional foods, due to their ability to be considered as matrices to encapsulate biologically active hydrophilic molecules. Both systems contained Medium Chain Triglycerides (MCT) as the continuous phase and were used as carriers of Hydroxytyrosol (HT), a hydrophilic antioxidant of olive oil. A low energy input fabrication process of the emulsion was implemented. The obtained emulsion contained 1.3% (w/w) of surfactants and 5% (w/w) aqueous phase. The spontaneously formed microemulsion contained 4.9% (w/w) of surfactants and 2% (w/w) aqueous phase. A comparative study in terms of structural characterization of the systems in the absence and presence of HT was carried out. Particle size distribution obtained by Dynamic Light Scattering (DLS) technique and interfacial properties of the surfactants' layer, examined by Electron Paramagnetic Resonance (EPR) spectroscopy indicated the involvement of HT in the surfactant membrane. Finally, the proposed systems were studied for the scavenging activity of the encapsulated antioxidant toward galvinoxyl stable free radical showing a high scavenging activity of HT in both systems.

Overexpression of caveolin-3-enhanced protein synthesis rather than proteolysis inhibition in C2C12 myoblasts: relationship with myostatin activity.

Caveolin-3 (cav-3), which is involved in the regulation of signal transduction and vesicular trafficking, could interact with activin receptor IIB to inhibit myostatin (MSTN) activity and may therefore play a role in muscle development and hypertrophy. MSTN is a member of the transforming growth factor-ß family, identified as a negative regulator of skeletal muscle mass. The expression of MSTN is fiber-type specific and the greatest amount of MSTN is present in fiber, which is composed of myosin heavy chain (MHC) type IIb. MSTN acts through the activin receptor IIB to activate smad2/3 which leads to an increase in gene transcription involved in muscle atrophy. Muscle hypertrophy is a consequence of two mechanisms: (1) the inhibition of proteolysis such as the calcium-dependent proteolytic system calpains and calpastatin and (2) an increase in protein synthesis through the Akt/mTOR/p70s6K pathway. In order to determine which of the two processes predominates in inhibition of MSTN activity in a cav-3 context, we transfected a C2C12 cell line with plasmids containing mstn or cav-3 wild genes. The results reported in this study demonstrate that inhibition of MSTN activity by overexpression of cav-3 induces an activation of protein synthesis rather than an inhibition of proteolysis through the calcium proteolytic system. The inhibition of phosphorylation of smad-3 due to overexpression of cav-3 causes an increase in the phosphorylation of the ribosomal protein S6, promoting the synthesis of MHC type II, probably through activation of Akt/mTOR/p70s6K. These data highlight the role of protein synthesis as the predominant mechanism in muscle hypertrophy observed when the expression of MSTN is altered and confirm the value of studying the physiological role of MSTN in the growing processes of skeletal muscle.

Coupling in vitro gastrointestinal lipolysis and Caco-2 cell cultures for testing the absorption of different food emulsions.

There is a growing interest in the optimization of dietary emulsions for monitoring postprandial lipid metabolism in the frame of preventing metabolic diseases. Using various emulsions, we investigated in a systematic scheme the combination of (i) in vitro gastrointestinal lipolysis and (ii) absorption and metabolism of lipolysis media in Caco-2 cells. Four emulsions based on either milk fat olein (OL) or rapeseed oil (RA) as the dispersed phase and either lecithin (LE) or sodium caseinate (CA) as the emulsifier were tested. After a sequential incubation of these emulsions with gastric and pancreatic enzymes, lipolysis media were incubated with Caco-2 cells, after dilution (1 : 20) to maintain the barrier integrity. Both gastric and duodenal lipolysis levels were similar to values reported in vivo and the rates of lipolysis were higher with LE-stabilized emulsions than with CA-stabilized emulsions (P < 0.05). TAG secretion by Caco-2 cells was found to be higher using (i) duodenal vs. gastric media (P < 0.001) and (ii) emulsions stabilized with CA vs. LE (P < 0.01). Consistently, gene expression of both FABP2 and FATP4 induced by the duodenal media was (i) higher than that with gastric media (P < 0.001) and (ii) faster than that with model mixed micelles. Using gastric media, TAG secretion of Caco-2 cells after 12 h was higher with RA than with OL (P < 0.001). Moreover, the RA-CA emulsion increased the size of secreted lipoprotein particles (514 nm vs. 61 to 130 nm; P < 0.01). In conclusion, it was possible to observe distinct responses in the lipid metabolism of Caco-2 cells incubated with lipolysis media obtained from different dietary emulsions digested by gastrointestinal lipases in vitro.

Influence of the oil globule fraction on the release rate profiles from multiple W/O/W emulsions.

Water-in-oil-in-water (W/O/W) double emulsions were prepared and the kinetics of release of magnesium ions from the internal to the external water phase was investigated as a function of the formulation and the globule volume fraction. All the emulsions were formulated using the same surface-active species (polyglycerol polyricinoleate and sodium caseinate). Also, the internal droplet and oil globule diameters were almost identical for all the systems. Two types of W/O/W emulsions were prepared based either on a synthetic oil (miglyol) or on an edible oil (olive oil). The globule volume fraction varied from 11% to 72%. At constant temperature (T=25 degrees C) and irrespective of the oil type, the percentage of magnesium released was lowered by increasing the globule fraction. In all cases, magnesium leakage occurred without film rupturing (no coalescence). Thus, the experimental data were interpreted within the frame of a model based on diffusion. The rate of release was determined by the permeation coefficient of magnesium across the oil phase and by the binding (chelation) of magnesium by caseinate molecules. The data could be adequately fitted by considering a time-dependant permeation coefficient. The better retention of magnesium at high globule fractions could account for two distinct phenomena: (i) the reduction of the relative volume of the outer phase, and (ii) the attenuation of the permeation coefficient over time induced by interfacial magnesium binding, all the more important than the globule fraction increased.