The role of oleosins and phosphatidylcholines on the membrane mechanics of oleosomes.

HYPOTHESIS: Oilseeds use triacylglycerides as main energy source, and pack them into highly stable droplets (oleosomes) to facilitate the triacylglycerides' long-term storage in the aqueous cytosol. To prevent the coalescence of oleosomes, they are stabilized by a phospholipid monolayer and unique surfactant-shaped proteins, called oleosins. In this study, we use state-of-the-art interfacial techniques to reveal the function of each component at the oleosome interface. EXPERIMENTS: We created model oil-water interfaces with pure oleosins, phosphatidylcholines, or mixtures of both components (ratios of 3:1, 1:1, 1:3), and applied large oscillatory dilatational deformations (LAOD). The obtained rheological response was analyzed with general stress decomposition (GSD) to get insights into the role of phospholipids and oleosins on the mechanics of the interface. FINDINGS: Oleosins formed viscoelastic solid interfacial films due to network formation via in-plane interactions. Between adsorbed phosphatidylcholines, weak interactions were observed, suggesting the surface stress response upon dilatational deformations was dominated by density changes. In mixtures with 3:1 and 1:1 oleosin-to-phosphatidylcholine ratios, oleosins dominated the interfacial mechanics and formed a network, while phosphatidylcholines contributed to interfacial tension reduction. At higher phosphatidylcholine concentrations (1:3 oleosin-to-phosphatidylcholine), phosphatidylcholine dominated the interface, and no network formation occurred. Our findings improve the understanding of both components' role for oleosomes.

Biosensing with Oleosin-Stabilized Liquid Crystal Droplets.

Liquid crystals (LCs) are emerging as novel platforms for chemical, physical, and biological sensing. They can be used to detect biological amphiphiles such as lipids, fatty acids, digestive surfactants, and bacterial endotoxins. However, designing LC-based sensors in a manner that preserves their sensitivity and responsiveness to these stimuli, and possibly improves biocompatibility, remains challenging. In this work, the stabilization of LC droplets by oleosins, plant-sourced and highly surface active proteins due to their extended amphipathic helix, is investigated. Purified oleosins, at sub-micromolar concentrations, are shown to readily stabilize nematic LC droplets without switching their alignment, allowing them to detect surfactants at micromolar concentrations. Direct evidence of localization of oleosins at the LC-water interface is provided with fluorescent labeling, and the stabilized droplets remain stable over months. Interestingly, chiral LC droplets readily switch in the presence of nanomolar oleosin concentrations, an unexpected behavior that is explained by accounting for the energy barriers required for switching the alignment between the two cases. This leads thus to a twofold conclusion: oleosin-stabilized nematic LC droplets present a biocompatible alternative for bioanalyte detection, while chiral LCs can be further investigated for use as highly sensitive sensors for detecting amphipathic helices in biological systems.

Insights into the emulsification mechanism of the surfactant-like protein oleosin.

Oleosins are proteins with a unique central hydrophobic hairpin designed to stabilize lipid droplets (oleosomes) in plant seeds. For efficient droplet stabilization, the hydrophobic hairpin with a strong affinity for the apolar droplet core is flanked by hydrophilic arms on each side. This gives oleosins a unique surfactant-like shape making them a very interesting protein. In this study, we tested if isolated oleosins retain their ability to stabilize oil-in-water emulsions, and investigated the underlying stabilization mechanism. Due to their surfactant-like shape, oleosins when dispersed in aqueous buffers associated to micelle-like nanoparticles with a size of ∼33 nm. These micelles, in turn, clustered into larger aggregates of up to 20 µm. Micelle aggregation was more extensive when oleosins lacked charge. During emulsification, oleosin micelles and micelle aggregates dissociated and mostly individual oleosins adsorbed on the oil droplet interface. Oleosins prevented the coalescence of the oil droplets and if sufficiently charged, droplet flocculation as well.

Gastrophysical and chemical characterization of structural changes in cooked squid mantle.

Squid (Loligo forbesii and Loligo vulgaris) mantles were cooked by sous vide cooking using different temperatures (46°C, 55°C, 77°C) and times (30 s, 2 min, 15 min, 1 h, 5 h, 24 h), including samples of raw tissue. Macroscopic textural properties were characterized by texture analysis (TA) conducted with Meullenet-Owens razor shear blade and compared to analysis results from differential scanning calorimetry. The collagen content of raw tissues of squid was quantified as amount of total hydroxyproline using ultra-high-performance liquid chromatography. Structural changes were monitored by Raman spectroscopy and small-angle X-ray scattering and visualized by second harmonic generation microscopy. Collagen in the squid tissue was found to be highest in arms (4.3% of total protein), then fins (3.0%), and lowest in the mantle (1.5%), the content of the mantle being very low compared to that of other species of squid. Collagen was found to be the major protein responsible for cooking loss, whereas both collagen and actin were found to be key to mechanical textural changes. A significant decreased amount of cooking loss was obtained using a lower cooking temperature of 55°C compared to 77°C, without yielding significant textural changes in most TA parameters, except for TA hardness which was significantly less reduced. An optimized sous vide cooking time and temperature around 55-77°C and 0.5-5 h deserves further investigation, preferably coupled to sensory consumer evaluation. PRACTICAL APPLICATION: The study provides knowledge about structural changes during sous vide cooking of squid mantle. The results may be translated into gastronomic use, promoting the use of an underutilized resource of delicious and nutritious protein (Loligo vulgaris and Loligo forbesii).

Evaluation of PBN spin-trapped radicals as early markers of lipid oxidation in mayonnaise.

Quality deterioration of mayonnaise is caused by lipid oxidation, mediated by radical reactions. Assessment of radicals would enable early lipid oxidation assessment and generate mechanistic insights. To monitor short-lived lipid-radicals, N-tert-butyl-α-phenylnitrone (PBN), a spin-trap, is commonly used. In this study, the fate of PBN-adducts and their impact on lipid oxidation mechanisms in mayonnaise were investigated. The main signals detected by Electron Spin Resonance (ESR) were attributed to L-radicals attached to 2-methyl-2-nitrosopropane (MNP), one of three degradation products of the PBN-peroxy-adduct. The second degradation product, benzaldehyde, was detected with Nuclear Magnetic Resonance (1H NMR), in line with MNP-L adduct generation. For the third class of degradation products, LO-radicals, their scission products were detected with 1H NMR and indicated that LO-radicals have a major impact on downstream oxidation pathways. This precludes mechanistical studies in presence of PBN. Degradation products of PBN-adducts can, however, be used for early assessment of antioxidants efficacy in oil-in-water emulsions.