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Water-in-oil microemulsions with biocompatible components were formulated to be used as carriers of natural antioxidants, such as hydroxytyrosol (HT) and gallic acid (GA). The system was composed of a mixture of natural surfactants, lecithin and monoglycerides, medium chain triglycerides, and aqueous phase. A dual approach was undertaken to study the structure and dynamics of these complicated systems. First, experimental data were collected by using adequate techniques, such as dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. Following this, a coarse-grained molecular dynamics (CGMD) study based on the experimental composition using the MARTINI force field was conducted. The simulations revealed the spontaneous formation of reverse micelles (RMs) starting from completely random initial conformations, underlying their enhanced thermodynamic stability. The location of the bioactive molecules, as well as the structure of the RM, were in accordance with the experimental findings. Furthermore, GA molecules were found to be located inside the water core, in contrast to the HT ones, which seem to lie at the surfactant interfacial layer. The difference in the antioxidants' molecular location was only revealed in detail from the computational analysis and explains the RM's swelling observed by GA in DLS measurements.

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Water-in-oil (W/O) microemulsions and emulsions based on medium chain triglycerides (MCT) were successfully formulated with the addition of emulsifiers and used as encapsulation matrices for hydroxytyrosol (HT), an antioxidant naturally found in extra virgin olive oil. The digestibility of these edible W/O dispersions by recombinant dog gastric lipase (rDGL) and porcine pancreatic lipase (PPL) was then tested at different pH values using a pHstat device. rDGL and PPL displayed a much lower activity on the W/O microemulsion than that on the W/O emulsion and MCT alone. This was explained by the presence of higher amounts of emulsifiers (4.9% w/w lecithin and monoglycerides) in the composition of W/O microemulsions compared to W/O emulsions (1.3% w/w emulsifiers). These surfactants also induced a shift of maximum lipase activity towards lower pH values, which usually reflects the competition between surfactants and lipases for binding at the lipid-water interface. rDGL and PPL were then used consecutively in a two-step digestion model mimicking the conditions found in the human gastrointestinal tract. Direct titration and back-titration of free fatty acids allowed the continuous estimation of lipolysis rates under both gastric and duodenal conditions. Gastric lipolysis of W/O microemulsions was reduced 6 to 9-fold compared to W/O emulsions. This inhibition had a major impact on the overall lipolysis, although duodenal lipolysis was less affected by the dispersion type. The presence of HT had also some minor effects on lipolysis rates.

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Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).

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